Over 350,000 adults die suddenly each year in the United States. The vast majority of adult sudden deaths are caused by atherosclerotic coronary artery disease and terminal ventricular arrhythmias. Although sudden unexplained deaths in the young are relatively uncommon, these deaths have a tremendous impact on both lay and medical communities. In children and young persons between the ages of 1 and 22 years, sudden unexplained deaths account for 11% of all deaths and claim over 4000 young persons each year. The incidence of sudden death in the young is between 1 and 5 per 100,000 patient-years (1). One of the first epidemiologic studies of sudden unexpected death in children and adolescents involved a review of death certificates of all residents of Olmsted County, Minnesota, who were between 1 and 22 years of age when they died during the period from January 1950 to October 1982 (2). Here, the incidence of sudden unexpected death was 1.3 per 100,000 patient-years.
Many sudden deaths in the young can be explained by cardiovascular abnormalities identifiable at autopsy, including myocarditis, hypertrophic cardiomyopathy, congenital coronary artery anomalies such as anomalous origin of left coronary artery from the right sinus of Valsalva, and aortic dissection (3). However, nearly half of these sudden deaths involve previously healthy children with no abnormal findings at autopsy. These deaths are dismissed as sudden unexplained death syndrome (SUDS). Thus, approximately 2000 sudden deaths in children are dismissed each year as SUDS. In fact, of the 12 individuals who died suddenly and unexpectedly in the Olmsted County population study, 6 of the 12 died of unknown causes, i.e., SUDS (2).
Previously, forensic pathologists could only speculate that a fatal arrhythmia might lie at the heart of SUDS because potentially lethal arrhythmogenic disorders like long QT syndrome (LQTS), Brugada syndrome, and Wolf-Parkinson-White syndrome leave no trace at autopsy examination. Clinically, LQTS is characterized by marked prolongation of the QT interval, and it can present with syncope, seizures, or sudden death. This syndrome may be the culprit in SUDS because its associated trademark arrhythmia, torsade de pointes, can be fatal. In England, there are 200 SUDS cases a year, and it is estimated that as many as one third of these deaths represent occult LQTS (3). However, as appealing as this diagnosis may be, without documentation of QT prolongation on an electrocardiogram (ECG) recorded before death, this concept of occult LQTS remains speculative.
Today, because of the molecular advances witnessed in the 1990s, LQTS constitutes the first genetically defined type of arrhythmia to be understood at the molecular level as caused by defective cardiac ion channels (4–6). To date, six LQTS loci, five LQTS genes, and more than 200 mutations have been identified in patients with LQTS (7–12). By probing these molecular targets, a new kind of forensic evaluation of SUDS involving a “molecular autopsy” is possible now. Here, we describe a case involving an adolescent boy discovered dead in bed. Using novel clinical testing in surviving family members and a molecular autopsy in the decedent, we established the cardiac channelopathy of LQTS as the cause and suggest a fatal arrhythmia as the manner of death.
MATERIALS AND METHODS
Case Report of Family With Sudden Unexplained Death
In August 1999, the decedent’s mother brought her 13-year-old son for evaluation in pediatric cardiology at the Mayo Clinic with this query: “Does my 13-year-old son have what killed my 17-year-old son 5 months ago?” The decedent had been found dead in bed in March 1999. The results of autopsy were unremarkable. The heart weighed 400 g, there was no myocyte disarray, and the coronary vessels were normal. The results of toxicology screening were negative except for caffeine. The cause and manner of death were not established. Nonetheless, it was believed in the community that a drug overdose must have occurred. An editorial in the local newspaper urged parents to talk to their children to prevent this kind of senseless tragedy attributable to drug use and abuse (13).
The decedent’s history was unremarkable. The family history was peculiar but inconclusive. His 13-year-old brother and father were asymptomatic. The decedent’s mother and maternal grandmother both described vague “waves of dizziness.” In addition, there were two unexplained accidents in childhood in the decedent’s mother. At age 9, she had fallen off the ladder to a 3-meter diving board. Reportedly, she landed on the cement, sustained no significant injury, and proceeded to climb up the ladder again and jump off the diving board with no sequelae. In the fourth grade, she apparently fell 20 feet from a fire escape without explanation. Both accidents were believed to be secondary to missteps. At the age of 34, she had a witnessed episode of syncope after same-day surgery approximately 4 hours after general anesthesia. The maternal grandmother reported three syncopal episodes. The first occurred in her 30s while she was watching the decedent’s mother have a plantar wart removed. She had a second episode of syncope 4 hours after she had shoveled snow. Her most recent episode occurred at age 60 while she was standing on the stairway. Several electrocardiograms were within normal limits, with no evidence of significant QT prolongation (i.e. QTc 460 milliseconds or more).
Clinical Evaluation of Sudden Unexplained Death Syndrome in Survivors
Standard Testing for Sudden Unexplained Death Syndrome in Survivors
Because of an unexplained death and a peculiar maternal family history, we screened the decedent’s brother and mother for LQTS with standard testing of 12-lead ECG, 24-hour ambulatory monitoring, and exercise stress testing. In addition, the brother had an echocardiogram. The echocardiogram was normal, and the LQTS screening test results were inconclusive.
Epinephrine Provocation Testing
An epinephrine provocation test was performed on the decedent’s mother despite the nondiagnostic standard evaluation. Baseline parameters of repolarization, including QT interval and rate corrected QT interval (QTc) as well as heart rate and blood pressure, were determined while the subject was in the supine position. Next, an epinephrine infusion was started at 0.1 μg/kg/min, and the same parameters were recorded. These measurements were made at 0.2 μg/kg/min epinephrine and at the highest infusion of 0.3 μg/kg/min. The subject was then monitored for 10 minutes of recovery with similar determinations.
Molecular Autopsy for LQTS Gene Defects
Written consent from the decedent’s mother and surviving first-degree relatives for this Mayo Foundation Institutional Review Board-approved proposal was obtained. Genomic DNA was extracted and isolated from a piece of paraffin-embedded myocardium using the QIAamp DNA Mini Kit (cat. No. 51306, Qiagen Inc., Valencia, CA, U.S.A.). In the surviving family members, DNA was extracted from peripheral blood lymphocytes using a standard phenol/chloroform extraction procedure (14). Genotyping of KVLQT1 was performed using the full-length genomic sequence and previously published intron/ exon-based primers (15). The mutation detection method involved exon-specific amplification by polymerase chain reaction and direct sequence analyses using manual ThermoSequenase sequencing (Amersham Life Science, Cleveland, OH) with 33P-labeled dideoxy nucleotide triphosphates, followed by single stranded conformation polymorphism gel analysis of relatives (16).
Standard Evaluation for Sudden Unexplained Death Syndrome
The results of standard clinical evaluation of the decedent’s immediate family were negative. Echocardiographic evaluation of the 13-year-old yielded normal results and no evidence of hypertrophic cardiomyopathy. The result of a review of several family members’ 12-lead electrocardiograms was equivocal. Figure 1 shows the mother’s screening ECG. The computer-generated QTc was 416 milliseconds, and this was corroborated manually. There were no abnormal T waves. Besides the decedent’s mother, the corrected QT intervals from several family members were 430 milliseconds (brother), 430 milliseconds (father), 450 milliseconds (maternal grandmother), 390 milliseconds (maternal grandfather), and 460 milliseconds (maternal aunt).
Despite a clinical evaluation indicating very low probability (Schwartz score = 1.5) for LQTS, the result of the mother’s epinephrine provocation study suggested this diagnosis (Figs. 2 and 3). Figure 2 demonstrates epinephrine-mediated changes in QT duration and T wave morphology. At rest, the absolute QT interval was 440 milliseconds. With infusion of 0.1 μg/kg/min epinephrine, the absolute QT interval paradoxically lengthened to 506 milliseconds (Fig. 3). With the concomitant increase in heart rate from 69 to 83 beats/min, the QTc increased from 450 to 575 milliseconds. In addition, the T wave morphology changed to a bifid, humped, M-shaped configuration at higher doses of epinephrine (Fig. 2).
Based upon the epinephrine-triggered alterations in repolarization, a molecular analysis of KVLQT1 was conducted. Figure 4 summarizes the characterization of the decedent’s LQTS-causing mutation. Direct DNA sequencing of the KVLQT1 gene identified a 5–base pair deletion in the polymerase chain reaction fragment containing exon 3 from genetic material isolated from the decedent’s paraffin-embedded heart tissue. The decedent was heterozygous for a mutation denoted as 735–739delGCGCT. This indicates that the 5 nucleotides: guanine (G), cytosine (C), guanine, cytosine, and thymidine (T) from nucleotide positions 735 through 739 are deleted. Translated to the cardiac potassium channel protein, this deletion causes a deleterious frame shift of amino acids 191 to 282, where a premature stop codon at amino acid 282 is introduced. The full-length channel contains 676 amino acids. This severely truncated mutant channel is missing 4 of the 6 critical transmembrane spanning domains and the essential ion channel pore (Fig. 4).
Figure 5 illustrates the decedent’s pedigree and the results of mutation-specific single stranded conformation polymorphism gel analysis. The heteroduplex band denotes the presence of the 5–base pair deletion. Despite equivocal screening QTc values, the defect was detected in the decedent’s brother, mother, maternal aunt, and maternal grandmother.
Indeed, the answer to the mother’s question—“Does my 13-year-old son have what killed my 17-year-old son?”—was yes. This case represents one of the first reported “molecular autopsy” diagnoses for SUDS. Previously, we described the postmortem molecular diagnosis of LQTS after an ultimately fatal near-drowning (17). This victim did manifest significant QT prolongation in the postresuscitation interval, providing some rationale to consider genetic LQTS. We successfully established the potential cause of death in a 12-year-old girl found dead in her room after extracting genetic material from autopsy tissue archived nearly 25 years previously (18). However, in retrospect, this case was not truly SUDS because the decedent had experienced two previous near-drownings and had ECG documentation of QT prolongation. This case, however, satisfies the criteria for sudden unexplained death syndrome: negative autopsy findings, no previous history, and no premortem ECG. Although the family history was peculiar, the family lacked any definitive evidence to invoke LQTS. In fact, the diagnostic criteria for inherited LQTS rendered a low-probability Schwartz score of 1.5 (19).
Here, traditional clinical tests used in the evaluation of SUDS survivors did not reveal LQTS. An epinephrine provocation study may assist in the sudden death evaluation by revealing “concealed” LQTS. The term concealed LQTS, or wrong QT syndrome, refers to an individual or family with molecular evidence of a LQTS channelopathy who does not manifest the hallmark finding of LQTS, namely, QT prolongation at rest. In general, a QTc of 480 milliseconds or more has a 100% positive predictive value, whereas a QTc of 400 milliseconds or less carries 100% negative predictive value (20). Unfortunately, as many as 30% of genetically proven LQTS subjects will possess an equivocal QTc between 400 and 480 milliseconds (21). These individuals are said to display “incomplete penetrance” of their LQTS genetic substrate. This equivocal zone of QTc values will also contain 5% to 10% of the normal population. Given the incidence of LQTS of 1:5000, for every 1000 subjects with an equivocal QTc (400–480), only 1 subject will be found to have LQTS. In an effort to minimize the number of false-positive and false-negative test results, most cardiologists use a QTc of 460 milliseconds or more as evidence of abnormal prolongation and a QTc of 420 to 460 milliseconds as borderline or equivocal (22).
It remains to be demonstrated whether or not epinephrine provocation will enhance the diagnostic accuracy of such subjects with borderline QT prolongation. In this family, all genotyped subjects have an incompletely penetrant LQTS substrate with resting QTc less than 460 milliseconds. The epinephrine provocation indicating paradoxical QT lengthening is distinct from the changes in QT parameters reported previously in nine healthy volunteers (23). In that study, the subjects displayed little change in the absolute QT interval. Because of the increased heart rate elicited during the epinephrine infusion, the subjects’ heart rate corrected QTc increased. In our study, the decedent’s mother lengthened not only the QTc but, importantly, the absolute QT interval as well. The decedent’s brother also showed this profile of paradoxical QT prolongation with epinephrine (data not shown). Further investigations are needed to determine whether this paradoxical QT response is a general maladaptive response in all LQTS subjects or is potentially pathognomonic for subjects with KVLQT1-based LQTS (LQT1).
Certainly, families with a history of an unexplained, premature sudden death warrant careful evaluation for heritable cardiovascular disorders. Although genotyping for cardiac ion channelopathies is unavailable currently as a routine clinical test, the ability to perform a molecular autopsy has transformed the forensic evaluation of unexplained sudden death in the young. Whether or not SUDS cases should be considered a primary cardiac channelopathy like LQTS until proved otherwise will require a determination of the molecular epidemiology of SUDS. Future studies are necessary to validate the suggestion that one third of SUDS are attributable to LQTS (3). If so, the postmortem molecular diagnosis may be lifesaving for other family members, because LQTS is quite treatable with beta-blocker therapy and/or implantable cardioverter defibrillator therapy (ICD). The mother and brother of this SUDS victim have an ICD.
In order to conduct these postmortem LQTS molecular analyses (“molecular autopsy”), the proper collection and storage of autopsy tissue is critical. Although we have succeeded in elucidating LQTS gene defects using paraffin-embedded tissues, this is not optimal starting material. Ideally, 10 to 15 ml of EDTA-blood and/or 5 to 10 g of heart, liver, or spleen tissue flash-frozen and stored at −80°C would provide the best source of genetic material to subject to gene(s)-wide analyses for defects. If the incidence of cardiac ion channel defects in SUDS is shown to be significant, there will be tremendous implications in terms of the forensic evaluation of SUDS, not to mention the clinical evaluation and counseling of SUDS survivors. Comprehensive evaluation of a decedent’s surviving family members may identify additional at-risk individuals before the family’s next tragedy.
The authors thank the family involved in this study and hope that the scientific lessons learned from their son’s death will one day prevent other families from experiencing a similar tragedy.
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