Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1 , KCNH2 , and SCN5A , and the 2 minor genes, KCNE1 and KCNE2 . Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value.
Recently, 2 studies have suggested that genomic rearrangements (large deletion/duplication mutations) involving KCNQ1 and KCNH2 may explain 5% to 12% of long QT syndrome (LQTS) in patients who were previously considered genotype negative after traditional open reading frame mutational analysis of the 3 canonical LQTS-causing genes: KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3). In addition, Caselli et al identified a 5.27-Mb deletion of chromosome 7 (7q36.1-q36.2) encompassing a whole gene deletion of KCNH2 in a 9-year-old with significant QT prolongation (corrected QT [QTc] duration 490 ms), renal hypoplasia, and mental retardation. Given these reports, we sought to determine the spectrum and prevalence of genomic rearrangements in our cohort of patients with LQTS who remained genetically elusive after conventional open reading frame mutation analysis of all 12 currently known LQTS-susceptibility genes (i.e., genotype-negative/phenotype-positive LQTS).
Methods
Our cohort consisted of 42 unrelated patients with robust clinical evidence for LQTS (QTc duration ≥480 ms and/or Schwartz-Moss score ≥4; Table 1 ). All patients were mutation negative after LQTS mutation analysis (denaturing high performance liquid chromatograph and sequencing) of the 3 canonical LQTS genes, KCNQ1 , KCNH2 , and SCN5A , and the 9 minor LQTS genes, ANKB , KCNE1 , KCNE2 , KCNJ2 , CACN1AC , CAV3 , SCN4B , AKAP9 , and SNTA1 . The study was approved by the Mayo Foundation institutional review board (Rochester, Minnesota).
| Male/female subjects | 14/28 |
| Average age (years) | 22.5 ± 15 |
| Average corrected QT duration | 530 ± 66 |
| Corrected QT duration ≥480 ms | 38 (90%) |
| Schwarz-Moss score ≥4 | 20 (48%) |
| Syncope or cardiac arrest | 21 (50%) |
| Family history of sudden cardiac death | 10 (24%) |
Genomic deoxyribonucleic acid was subjected to multiplex ligation-dependent probe amplification (MLPA) analysis, a quantitative technique for detecting large deletions and duplications otherwise missed by traditional mutation detection methods, using the SALSA P114 LQT kit (MRC-Holland, Amsterdam, The Netherlands) according to manufacturer’s instructions. Data were collected and analyzed with GeneMarker 1.75 (Soft Genetics, State College, Pennsylvania). Significantly (>35%) decreased or increased signal relative to control probes were scored as exon deletions or duplications, respectively.
To confirm and decipher the deletion breakpoints in KCNQ1 , long-range polymerase chain reaction (PCR) amplification using the Expand Long Template PCR System (Roche, Mannheim, Germany) and PCR primers flanking the suspected boundaries of the deletion was performed. The amplicon was isolated on agarose gel electrophoresis, excised from the gel, purified using the QIAquick gel extraction kit (Qiagen Sciences, Germantown, Maryland), and deoxyribonucleic acid sequenced. Primer sequences and PCR conditions are available upon request.
Results
After MLPA analysis, 2 unique KCNQ1 gene rearrangements were identified in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11) with genotype-negative/phenotype-positive LQTS. A 5,306-basepair (bp) deletion (c.478-5001_604 + 178del; Figure 1 ) of KCNQ1 ( NM_000218.2 ), involving a portion of intron 2 (5,001 bp), all of exon 3 (127 bp), and a portion of intron 3 (178 bp) that results in the complete deletion of exon 3 and a subsequent frameshift of the KCNQ1 transcript leading to a premature stop codon (p.E160fs34X; Figure 1 ), was identified in a 10-year-old Caucasian boy. This patient was diagnosed clinically at 5 years of age with extreme QT prolongation (QTc duration 660 ms), recurrent seizures with activity and flashing lights since 2 years of age (normal electroencephalogram), a personal history of 5 syncopal episodes during physical activity, and, although atypical of LQTS, a family history of syncope associated with onset of pain in his mother and maternal grandmother. The patient has been deaf since infancy, which may be related to viral meningitis occurring at 2 months of age. At 10 years of age, he received an implantable cardioverter–defibrillator after breakthrough syncope during exertion while on propranolol β-blocker therapy. Six weeks after device implantation, the patient received 16 appropriate shocks on 1 day after multiple episodes of torsade de pointes. Samples from affected relatives were unavailable to confirm proper pedigree cosegregation of the mutation.
Deletions of 4,595 bp (portion of intron 6, c.922-7066_922-2471del; Figure 2 ) and 4,122 bp (c.922-2359_1032 + 1652del; Figure 2 ) of KCNQ1 involving a portion of intron 6 (2,359 bp), all of exon 7 (111 bp), and all of intron 7 (1,652 bp) except for 14 bp leading up to exon 8 were identified in a 17-year-old Caucasian girl. These deletions presumably result in a complete deletion of exon 7 and exon skipping of exon 8 due to abnormal splicing resulting from loss of the putative branchpoint sequence of intron 7 ( Figure 2 ). This patient had a QTc duration of 480 ms with lengthening to 515 ms during exercise stress testing, a personal history of 2 syncopal episodes during physical activity at 6 years of age, and a family history of documented QT prolongation during exercise stress testing in a 13-year-old brother (QTc duration 428 ms at rest lengthening to 573 ms during exercise), abnormal T waves in a 19-year-old sister (QTc duration 454 ms), and a clinical diagnosis of LQTS in the mother.
All 3 family members with clinically suspected LQTS are asymptomatic and being treated with nadolol. In addition, 2 brothers (16 years old with QTc duration 400 ms and asymptomatic and 12 years old with QTc duration 424 ms and a history of “neurocardiogenic” syncope 1 time while going to the rest room and 2 times while standing in the heat) have been diagnosed clinically as normal and are untreated. Pedigree mutational analysis ( Figure 2 ) confirmed that all clinically diagnosed relatives indeed hosted the mutation. In addition, 1 of the siblings dismissed as “normal” is genotype positive and preventative measures (QT drug avoidance) for his concealed LQT1 have been implemented.
Results
After MLPA analysis, 2 unique KCNQ1 gene rearrangements were identified in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11) with genotype-negative/phenotype-positive LQTS. A 5,306-basepair (bp) deletion (c.478-5001_604 + 178del; Figure 1 ) of KCNQ1 ( NM_000218.2 ), involving a portion of intron 2 (5,001 bp), all of exon 3 (127 bp), and a portion of intron 3 (178 bp) that results in the complete deletion of exon 3 and a subsequent frameshift of the KCNQ1 transcript leading to a premature stop codon (p.E160fs34X; Figure 1 ), was identified in a 10-year-old Caucasian boy. This patient was diagnosed clinically at 5 years of age with extreme QT prolongation (QTc duration 660 ms), recurrent seizures with activity and flashing lights since 2 years of age (normal electroencephalogram), a personal history of 5 syncopal episodes during physical activity, and, although atypical of LQTS, a family history of syncope associated with onset of pain in his mother and maternal grandmother. The patient has been deaf since infancy, which may be related to viral meningitis occurring at 2 months of age. At 10 years of age, he received an implantable cardioverter–defibrillator after breakthrough syncope during exertion while on propranolol β-blocker therapy. Six weeks after device implantation, the patient received 16 appropriate shocks on 1 day after multiple episodes of torsade de pointes. Samples from affected relatives were unavailable to confirm proper pedigree cosegregation of the mutation.
