Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin-2 (PKP2) identified with microarray analysis and/or multiplex ligation-dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.

Cardiac risk assessment before the use of stimulant medications in children and youth: A joint position statement by the Canadian Paediatric Society, the Canadian Cardiovascular Society, and the Canadian Academy of Child and Adolescent Psychiatry.

Regulatory decisions and scientific statements regarding the management of attention-deficit hyperactivity disorder (ADHD) raise questions about the safety of medications and the appropriate pretreatment evaluation to determine suitability for treatment with medication. This is particularly true in the setting of known structural or functional heart disease. The present paper reviews the available data, including peer-reviewed literature, data from the United States Food and Drug Administration Web site on reported adverse reactions in children using stimulant medication, and Health Canada data on the same problem. A consensus-based guideline on appropriate assessment is provided, based on input from members of the Canadian Paediatric Society, the Canadian Cardiovascular Society and the Canadian Academy of Child and Adolescent Psychiatry, with specific expertise and knowledge in the areas of both ADHD and pediatric cardiology. The present statement advocates a thorough history and physical examination before starting stimulant medications, with an emphasis on the identification of risk factors for sudden death, but does not routinely recommend electrocardiographic screening or cardiac subspecialist consultation unless indicated by history or physical examination findings. A checklist for identifying children who are potentially at risk of sudden death (independent of ADHD or medications used to treat it) is provided. Although recommendations are based on the best evidence currently available, the committee further agrees that more research on this subject is necessary to optimize the approach to this common clinical scenario.

Diagnosis of postoperative arrhythmias following paediatric cardiac surgery.

Arrhythmias are commonly encountered in the paediatric intensive care unit setting, most frequently in the setting of postoperative congenital heart disease. Postoperative arrhythmias are an important cause of morbidity in children in the postoperative period following cardiac surgery for congenital cardiac lesions. It is important for all paediatric critical care physicians involved in the care of these children to understand the potential mechanisms involved and how to make an accurate diagnosis. The existing literature has focused on small groups and specific arrhythmias. There is a paucity of literature to guide the clinician in approaching arrhythmias in the paediatric intensive care unit setting. Our objective was to review the recognition and diagnosis of paediatric arrhythmias in the postoperative period following congenital cardiac surgery. Timely and accurate identification of the rhythm disturbance is mandatory and allows for the institution of effective, rhythm specific management strategies.

Cardiac risk assessment before the use of stimulant medications in children and youth.

Regulatory decisions and scientific statements regarding the management of attention-deficit hyperactivity disorder (ADHD) raise questions about the safety of medications and the appropriate pretreatment evaluation to determine suitability for treatment with medication. This is particularly true in the setting of known structural or functional heart disease. The present paper reviews the available data, including peer-reviewed literature, data from the United States Food and Drug Administration Web site on reported adverse reactions in children using stimulant medication, and Health Canada data on the same problem. A consensus-based guideline on appropriate assessment is provided, based on input from members of the Canadian Paediatric Society, the Canadian Cardiovascular Society and the Canadian Academy of Child and Adolescent Psychiatry, with specific expertise and knowledge in the areas of both ADHD and paediatric cardiology. The present statement advocates a thorough history and physical examination before starting stimulant medications, with an emphasis on the identification of risk factors for sudden death, but does not routinely recommend electrocardiographic screening or cardiac sub-specialist consultation unless indicated by history or physical examination findings. A checklist for identifying children who are potentially at risk of sudden death (independent of ADHD or medications used to treat it) is provided. Although recommendations are based on the best evidence currently available, the committee further agrees that more research on this subject is necessary to optimize the approach to this common clinical scenario.

Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control.

AIMS/HYPOTHESIS: Activation of protein kinase C (PKC) isoforms has been implicated as a central mediator in the pathogenesis of diabetic nephropathy. Although high glucose levels stimulate catalytic activity of PKC, the effects of high glucose levels on the expression of genes encoding PKC isoforms are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta. METHODS: Recent advances in molecular biological techniques now permit quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed. RESULTS: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies of diabetic patients relative to control (p<0.001). No increase in PRKC-alpha expression was seen. In addition, a correlation between renal PRKC-beta mRNA and HbA(1c) was observed in diabetic patients (r=0.63, p<0.05). There was co-localisation of PKC-beta and phospho-PKC-beta predominantly to proximal tubules. A 60% increase in PRKC-beta mRNA and peptide in cultured human proximal tubular epithelial cells exposed to high glucose (p<0.05) was seen in vitro. CONCLUSIONS/INTERPRETATION: PKC-beta is upregulated at the gene expression level in human diabetic nephropathy. PRKC-beta mRNA correlates closely with serum HbA(1c), possibly partially explaining the relationship between glycaemic control and progression of diabetic nephropathy. Archival human tissue provides a valuable resource for molecular analyses.

Clinical use of permanent pacemaker for conversion of intraatrial reentry tachycardia in children.

The use of the implanted atrial-based pacemaker to overdrive postsurgical intraatrial reentry tachycardia (IART) was evaluated in a large group of pediatric patients over a 14-year study period. The authors sought to determine the feasibility of this noninvasive technique in the management of this specialized population and to determine factors associated with successful conversion. They examined 128 manual overdrive attempts performed on 22 consecutive patients. There were 10 patients with post-Fontan repair, 7 with post-Mustard/Senning procedure, and 5 with miscellaneous lesion types. The number of IART episodes for overdrive pacing per patient ranged from 1 to 15. The first overdrive pacing attempt was successful in 63% (14/22) of the patients. The mean IART cycle length was 278 +/- 59 ms. The mean pacing rate for effective conversion of IART was 66 +/- 10% faster than the IART rate. By controlling for repeated measures for individual patients, three factors were found to be independently associated with a successful outcome: (1) lesion type other than Fontan surgery (P = 0.007), (2) lack of acceleration of IART with the overdrive attempt (P < 0.001), and (3) patient use of amiodarone with attempt (P = 0.005). There were three procedural complications: two inadvertent overdrive pacing episodes, and one episode of acceleration of IART cycle length and conduction resulting in need for cardioversion. Manual pacemaker overdrive conversion of IART is a useful adjunct in the management of postsurgical IART in the pediatric population and should be considered as an initial treatment option.

Exercise capacity in children with hypertrophic cardiomyopathy and its relation to diastolic left ventricular function.

Left ventricular diastolic impairment is often seen in children with hypertrophic cardiomyopathy regardless of left ventricular outflow tract obstruction. Such impairment in diastolic filling is related to the presence of symptoms and exercise impairment.

Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats.

A single intraperitoneal injection of lipopolysaccharide (LPS) causes a biphasic suppression of testicular steroidogenesis in adult rats, with inhibition at 6 h and 18-24 h after injection. The inhibition of steroidogenesis is independent of the reduction in circulating LH that also occurs after LPS treatment, indicating a direct effect of inflammation at the Leydig cell level. The relative contributions to this inhibition by intratesticular versus systemic responses to inflammation, including the adrenal glucocorticoids, was investigated in this study. Adult male Wistar rats (eight/group) received injections of LPS (0.1 mg/kg i.p.), dexamethasone (DEX; 50 microg/kg i.p.), LPS and DEX, or saline only (controls), and were killed 6 h, 18 h and 72 h later. Treatment with LPS stimulated body temperature and serum corticosterone levels measured 6 h later. Administration of DEX had no effect on body temperature, but suppressed serum corticosterone levels. At the dose used in this study, DEX alone had no effect on serum LH or testosterone at any time-point. Expression of mRNA for interleukin-1beta (IL-1beta), the principal inflammatory cytokine, was increased in both testis and liver of LPS-treated rats. Serum LH and testosterone levels were considerably reduced at 6 h and 18 h after LPS treatment, and had not completely recovered by 72 h. At 6 h after injection, DEX inhibited basal IL-1beta expression and the LPS-induced increase of IL-1beta mRNA levels in the liver, but had no effect on IL-1beta in the testis. The effects of DEX on IL-1beta levels in the liver were no longer evident by 18 h. In LPS-treated rats, DEX caused a significant reversal of the inhibition of serum LH and testosterone at 18 h, although not at 6 h or 72 h. Accordingly, DEX inhibited the systemic inflammatory response, but had no direct effect on either testicular steroidogenesis or intra-testicular inflammation, at the dose employed. These data suggest that the inhibition of Leydig cell steroidogenesis at 6 h after LPS injection, which was not prevented by co-administration of DEX, is most likely due to direct actions of LPS at the testicular level. In contrast, the later Leydig cell inhibition (at 18 h) may be attributable to extra-testicular effects of LPS, such as increased circulating inflammatory mediators or the release of endogenous glucocorticoids, that were inhibited by DEX treatment. These data indicate that the early and late phases of Leydig cell inhibition following LPS administration are due to separate mechanisms.

Mortality following radiofrequency catheter ablation (from the Pediatric Radiofrequency Ablation Registry). Participating members of the Pediatric Electrophysiology Society.

Deaths have been reported following radiofrequency catheter ablation (RFCA), but the mortality rate in children has not been defined. This study sought to analyze the incidence and the factors associated with mortality related to RFCA. Ten of 4,651 cases (0.22%) reported to the Pediatric RFCA Registry resulting in death were reviewed and compared with a matched control group (n = 18). Death occurred in 5 of 4,092 children (0.12%, ages 0.1 to 13.3 years) with structurally normal hearts. Death was related to traumatic injury, myocardial perforation and hemopericardium, coronary or cerebral thromboembolism, and ventricular arrhythmia. All cases were left-sided (p = 0.019 vs right or septal) supraventricular arrhythmias with radiofrequency applications in the systemic atrium and/or ventricle, and all procedures were successful. Mortality occurred in 5 of 559 children (0.89%, p = 0.001 vs normals, ages 1.5 to 17.4 years) with structural heart disease. No new pathology except the mural radiofrequency lesions was seen at autopsy. Those with structurally normal hearts who died were smaller (32.7 vs 55.6 kg, p = 0.023) and had more radiofrequency applications (26.3 vs 8.7, p = 0.019) than those who survived. No differences were demonstrated for those with abnormal hearts. Operator experience was not different (deaths 103 +/- 106 vs controls 117 +/- 125, p = 0.41). Mortality associated with pediatric RFCA is rare, but is more frequent when there is underlying heart disease, lower patient weight, greater number of radiofrequency energy applications, and left-sided procedures. Operator experience does not appear to be a factor leading to mortality.

Propafenone disposition during continuous venovenous hemofiltration.

OBJECTIVE: To determine the extent of removal of propafenone by continuous venovenous hemofiltration (CVVH) in a critically ill pediatric patient. CASE SUMMARY: A three-year-old white-Japanese girl was admitted to the critical care unit following cardiac surgery. Her postoperative course was complicated by the development of junctional ectopic tachycardia, requiring propafenone, and acute renal failure, which necessitated the use of CVVH. The serum and ultrafiltrate concentrations of propafenone and its 5-hydroxy metabolite were measured to determine both total and CVVH clearance. CONCLUSIONS: The data from this case report showed that propafenone was not significantly removed by CVVH. Furthermore, the total clearance of propafenone was not affected by the patient's renal or liver function impairment.

Accuracy of surface electrocardiograms for differentiating children with hypertrophic cardiomyopathy from normal children.

Most patients with hypertrophic cardiomyopathy have abnormal electrocardiograms. In this study of 37 matched pairs in the pediatric age group, the 12-lead electrocardiogram did not differentiate between affected and normal children reliably enough to allow it to be used as a screening test in the general population.

Chronotropic competence of the sinus node in congenital complete heart block.

Electrocardiograms taken at rest of 2 children with transplacental exposure to anti-Ro antibody but 1:1 atrioventricular conduction demonstrated sinus node disease. Treadmill exercise testing of 28 patients with congenital complete heart block found 3 patients with chronotropic incompetence of the sinus node.

Total cavopulmonary anastomosis (Fontan) in children with Down's syndrome.

BACKGROUND: There is a paucity of information to guide the management of the child with Down's syndrome and congenital heart disease in whom biventricular repair is precluded. METHODS: Through the cardiology and cardiovascular surgery databases of The Hospital for Sick Children and Toronto Congenital Cardiac Centre for Adults, we identified patients with trisomy 21 and ventricular hypoplasia who had undergone a Fontan procedure (or modification). RESULTS: Of 533 patients who had undergone a Fontan operation between 1976 and 1997, 4 had trisomy 21. All 4 patients had unbalanced complete atrioventricular septal defect with right ventricular hypoplasia in 3 and left ventricular hypoplasia in 1. Three patients survived, and 1 died of endocarditis. The 3 survivors have done well in the short term and medium term without complications related to the pulmonary vasculature. CONCLUSIONS: We suggest that in appropriately selected patients with trisomy 21 and ventricular hypoplasia who are unsuitable for two or one and a half ventricle repair, the Fontan procedure is not contraindicated and provides short-term and medium-term benefit.

Obstructive rhabdomyoma and univentricular physiology: a rare combination.

We report the successful excision of a large left atrial rhabdomyoma producing complete obstruction of both inflow and outflow to the left ventricle. Systemic perfusion was dependent on anterograde ductual flow. The resultant univentricular physiology was initially managed medically, with spontaneous tumor regression contemplated as a means of possible long-term "cure." Failure to achieve hemodynamic stability compelled urgent surgical excision. This neonate was successfully discharged home with an in-series biventricular circulation.

Impact of programmed sensitivity safety factor on atrial sensing in children.

The relationship between the pacemaker sensitivity safety factor (PSSF) and atrial under- or oversensing as documented by 24-hour Holter monitoring was examined. Our study comprised 78 transvenous fixed atrial leads implanted between 1983-1995 in 71 children. Overall, 210 Holter reports identified 143 (68%) Holters with normal atrial sensing function, 31 (15%) with undersensing, 32 (15%) with oversensing, and 4 (2%) with both problems. From 161 Holter reports in which the PSSF was available, the incidence of undersensing at a PSSF of 2.0 (range 1.5-2.4) was 25% (14/57). There was a dramatic decline in undersensing when the PSSF was > or = 3 (3%) compared to a PSSF < 3 (21%) (P < 0.001). A PSSF cut-off point of 2.0 best predicted occurrence of undersensing with a sensitivity of 79% and a specificity of 67%. Other variable were also examined by multiple logistic regression analysis, but only PSSF remained highly associated with undersensing (odds ratio [OR] = 0.6, P = 0.03). In contrast, PSSF did not have a significant role in predicting oversensing, but presence of sick sinus syndrome (OR = 10.5) or unipolar lead (OR = 5.6) were significantly associated with oversensing (P = 0.0001). The majority of undersensing problems can be avoided by routinely allowing for at least a threefold or more programmed sensitivity margin. Other factors may increase the risk of oversensing, regardless of the PSSF.