Jason Y. Park

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Review article: the global emergence of Helicobacter pylori antibiotic resistance

Helicobacter pylori is one of the most prevalent global pathogens and can lead to gastrointestinal disease including peptic ulcers, gastric marginal zone lymphoma and gastric carcinoma.

Guanylyl Cyclase C Messenger RNA Is a Biomarker for Recurrent Stage II Colorectal Cancer

Stage at diagnosis is the most important prognostic determinant for patients with colorectal cancer (1-6), and it dictates the role of adjuvant chemotherapy in this disease (7-10). Given the prognostic and therapeutic importance of staging, accurate histopathologic evaluation of lymph nodes to detect invasion by tumor cells is crucial. However, conventional microscopic lymph node examination has methodologic limitations (6, 11). It can be difficult to differentiate single or even small clumps of tumor cells from other types of cells, which limits sensitivity. The standard practice of examining only a limited number of tissue sections from each lymph node can omit from review more than 99% of each specimen and can introduce sampling error. These limitations are evident when the frequency of disease recurrence in patients with stage I and stage II disease is considered. By definition, such patients do not have extraintestinal disease at the time of curative resection. However, recurrence rates of 10% to 30% have been reported for lesions confined to the mucosa (stage I disease), and rates of 30% to 50% have been reported for lesions confined to the bowel wall (stage II disease) (12, 13). Alternate methods of detecting small numbers of tumor cells have been used for staging, including intensive review of serial tissue sections, immunohistochemical analysis to detect tumor-associated antigens, polymerase chain reaction (PCR) to detect tumor-specific mutations, and reverse transcriptase (RT) PCR to detect the expression of tumor-associated biomarkers (6, 11). In some studies of colorectal cancer, staging by these sensitive methods has been correlated with disease. However, the fact that serial sectioning is labor- and cost-intensive, the lack of uniform association between mutations and neoplastic transformation, and the nonspecificity of many biomarkers limit the applicability of these methods. An easily detected biomarker that is specifically expressed by all colorectal tumors would be useful for disease staging. Guanylyl cyclase C is expressed in normal intestinal mucosal cells, adenomatous polyps, and primary and metastatic colorectal tumors but not in extraintestinal tissues or tumors (14-17). Expression of guanylyl cyclase C has been detected by RT-PCR in all of the histologically confirmed colorectal tumors and colorectal cancer cell lines that have been examined (14-17). Therefore, guanylyl cyclase C may be a specific biomarker for metastasis of extraintestinal colorectal cancer (16, 17). We examined whether expression of guanylyl cyclase C messenger RNA (mRNA) in lymph nodes was associated with disease recurrence in patients with stage II colorectal cancer who had presumably been cured by surgical resection. Methods Patients and Tissues We examined the tumor registry database at Thomas Jefferson University (Philadelphia, Pennsylvania) for patients who had been treated for colorectal cancer between 1989 and 1995, an interval that permitted adequate follow-up (Figure). The initial search was designed to exclude patients who developed recurrent disease more than 3 years after the index surgery. In this way, we avoided inadvertent inclusion of patients who had metachronous rather than recurrent cancer. Our search yielded 445 patients with invasive colon or rectal cancer and no evidence of metastases (tumor, node, metastasis [TNM] classification, N0 M0) at surgery. Of these 445 patients, 260 had surgery at Thomas Jefferson University that yielded lymph nodes. Subsequently, 167 patients were excluded because they had stage I or less severe disease (T0-T2 N0 M0), because they developed recurrent disease locally or at unspecified sites, or because they received neoadjuvant chemotherapy or radiation therapy. Fifty-six patients with no evidence of recurrence were excluded because they had less than 6 years of follow-up. Of 18 patients who had had no evidence of disease for 6 or more years after surgery and were considered clinically cured, 16 had pathologic specimens available for further analysis; these patients formed the control group. Of 19 patients who developed metastases up to 3 years after surgery, 12 had pathologic specimens available for further analysis; these patients formed the case-patient group. The remaining 9 patients were excluded from analysis. Two controls (12.5% [patients 9 and 16]) and 1 case-patient (8.3% [patient 24]) received 5-fluorouracil-based adjuvant chemotherapy after surgery. Figure. Algorithm for selecting patient biopsy samples for analysis. Reverse Transcriptase Polymerase Chain Reaction In our study, lymph nodes were obtained for analysis under an institutional review board-approved protocol that maintained patient anonymity. Preliminary tests showed that mRNA isolated from single 10-m sections of individual lymph nodes yielded insufficient RNA for RT-PCR analysis. Consequently, at least five 10-m sections of representative lymph nodes for each patient were pooled and deparaffinized, and the total RNA was isolated (17). Reverse transcriptase polymerase chain reaction was performed by using the RNA PCR Kit, version 2 (Takara Shuzo Co., Ltd., Kyoto, Japan) (16, 17). We used only total RNA that yielded amplicons after -actin-specific RT-PCR was used (16, 17). Guanylyl cyclase C-specific RT-PCR and nested carcinoembryonic antigen-specific RT-PCR were performed as described elsewhere (16-18). Reactions from RT-PCR were separated by electrophoresis on 4% NuSieve 3:1 agarose (FMC Bioproducts, Rockland, Maine) and by amplification products visualized by ethidium bromide. We included positive control specimens, consisting of RNA isolated from human colorectal cancer cells that expressed guanylyl cyclase C and carcinoembryonic antigen (Caco2 cells [American Type Culture Collection, Rockville, Maryland]), and negative control specimens, consisting of RNA from lymph nodes without colorectal cancer and incubations in which no template was added. Amplicon identity was confirmed by sequencing (16, 17). Production of guanylyl cyclase C-specific amplicons was confirmed by Southern blot analysis, which used a 32P-labeled antisense probe that complemented a sequence found within the amplicon (19). Statistical Analysis Results are expressed as the mean SD, except for disease-free and overall survival, which are expressed as the median (range). We calculated P values by using the Fisher exact test. The odds ratios, with exact 95% CIs, were calculated by using the StatXact 4.0 statistical software package (Cytel Software Corp., Cambridge, Massachusetts). Role of the Funding Source Targeted Diagnostics and Therapeutics, Inc., which provided a portion of the grant support for this study, was not involved in the design of the study or in the collection and analysis of the data; it also had no role in the decision to submit the paper for publication. Results Characteristics of Patients Evaluated by Reverse Transcriptase Polymerase Chain Reaction Patients ranged in age from 37 to 85 years (mean, 68.1 9.5 years). Women and men were similar in age (range, 52 to 85 years [mean, 64.5 10.5 years] and 37 to 82 years [mean, 70.9 7.8 years], respectively). The ratio of men to women was 8:9 among controls and 5:7 among case-patients. One woman was African-American; all other patients were white. The ratio of cases of T3 to T4 disease was 3:13 among controls and 4:8 among case-patients. Patients were followed for 9 to 105 months (mean, 67.4 30.7 months). Controls were followed for 73 to 105 months (mean, 89.9 7.8 months), and case-patients were followed for 9 to 78 months (mean, 37.3 22.6 months). In the control group, 1 patient (6.3%) developed a new primary colonic lesion 96 months after initial diagnosis, 1 patient (6.3%) died of causes unrelated to colorectal cancer, and the remaining 14 patients (87.5%) were alive and free of disease 88 months after diagnosis (range, 73 to 97 months). In the case-patient group, 8 patients (66.7%) died of recurrent colorectal cancer after 13 months of disease-free survival (range, 3 to 35 months) and after 19 months of overall survival (range, 9 to 64 months). Four patients (33%) were alive with metastases after 12 months of disease-free survival (range, 2 to 36 months) and 52 months of overall survival (range, 17 to 78 months). Analysis by Reverse Transcriptase Polymerase Chain Reaction of RNA Expression in Lymph Nodes For all 28 patients, 524 lymph nodes (mean, 18.4 12.5 lymph nodes per patient) collected at surgery were reported to be free of tumor in the original histologic review. The number of lymph nodes obtained from each patient at the time of initial operative staging was similar in the control group (mean, 19.9 13.2 lymph nodes per patient) and the case-patient group (mean, 17.2 12.7 lymph nodes per person). Lymph nodes were omitted from RT-PCR analysis because they were not available from the pathology department (326 lymph nodes from 28 patients [62.2% of 524 lymph nodes obtained at surgery]). Of the 198 lymph nodes that were available for RT-PCR analysis, 19.7% (39 lymph nodes from 7 patients [7.4% of 524 lymph nodes obtained at surgery]) did not yield RNA. The number of lymph nodes available for RT-PCR analysis was similar in the control group (mean, 6.4 3.0 lymph nodes) and the case-patient group (mean, 8.1 6.3 lymph nodes). Twenty-one patients (75%) yielded 159 paraffin-embedded lymph nodes (mean, 7.6 5.2 lymph nodes per patient) that could be adequately evaluated by RT-PCR. In 5 case-patients (41.7%) and 2 controls (16.7%), -actin-specific amplicons (an indicator of intact RNA) were not detected in the total RNA from pooled sections of lymph nodes; these 7 patients were excluded from further analysis. Total RNA extracted from the pooled lymph node sections of the remaining 21 patients was analyzed by RT-PCR using guanylyl cyclase C-specific primers. Guanylyl cyclase C-specific amplicons were not detected in any reaction that used RNA from lymph nodes of control

Molecular Signatures of Pancreatic Cancer

CONTEXT The introduction of genome- and epigenome-wide screening techniques has dramatically improved our understanding of the molecular mechanisms underlying the development of pancreatic cancer. There are now 3 recognized histologic precursors of pancreatic cancer: pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and mucinous cystic neoplasm. Each of these precursor lesions is associated with specific molecular alterations. OBJECTIVE To understand the molecular characteristics of pancreatic ductal adenocarcinoma and its precursor lesions. DATA SOURCES PubMed (US National Library of Medicine). CONCLUSIONS In this review, we briefly summarize recent research findings on the genetics and epigenetics of pancreatic cancer. In addition, we characterize these molecular alterations in the context of the histologic subtypes of pancreatic cancer.

Mycophenolic Acid (cellcept and Myofortic) Induced Injury of the Upper Gi Tract

Background Mycophenolic acid (MPA) is an immunosuppressant drug commonly used in patients undergoing solid organ transplant. Although its pattern of inducing injury in the colon is well-known and features prominent crypt apoptosis that mimics graft-versus-host-disease, the injury pattern in the upper gastrointestinal (GI) tract is less extensively documented. We studied the pattern of upper GI tract injury in symptomatic patients taking MPA. Design Twenty solid organ transplant patients who were taking MPA and had concurrent upper GI tract biopsies were identified on a laboratory information system search. From these 20 patients, 17 duodenal, 18 gastric, and 7 esophageal biopsies were examined. All patients were symptomatic. Apoptosis and patterns of chronic and active injuries were assessed on standard hematoxylin and eosin, periodic acid-Schiff/Alcian blue, and Diff-Quik stained slides. To measure the significance of apoptosis, we standardized the apoptotic counts in normal biopsies using duodenal, gastric, and esophageal biopsies from 45 normal cases and performed statistical analysis. For the purposes of this study, we regarded apoptotic counts higher than the mean plus 2 SDs as significant. Thus the cut-off values for apoptosis were ≥2 apoptotic bodies/100 crypts for duodenum, ≥3/100 glands for stomach, and ≥2/10 high-power field for esophagus. Results GI-related symptoms and abnormalities manifested between 1 month and 10 years posttransplant and included diarrhea (55%); nausea (45%); abdominal pain (35%); vomiting (25%); GI bleeding (15%); dysphagia (10%); dyspepsia, anemia, and hematemesis (5% for each). Most (14 out of 17, 82%) duodenal biopsies showed apoptotic counts of ≥2/100 crypts, 28% (5 out of 18) of gastric biopsies showed apoptotic counts of ≥3/100 glands, and 57% (4 out of 7) of esophageal biopsies showed apoptotic counts of ≥2/10 high-power field. Four gastric biopsies showed a previously undescribed injury pattern of parietal cells resembling the ballooning degeneration. Additional pathologic findings included: chronic peptic duodenitis (6 out of 17, 35%), active duodenitis (1 out of 17, 6%), and celiac-like features (2 out of 17, 12%) in the duodenum; chemical gastropathy (3 out of 18, 17%), active chronic gastritis without Helicobacter pylori (2 out of 18, 11%), and erosion (1/18,6%) in the stomach; reactive epithelial change (3 out of 7, 43%), active esophagitis (3 out of 7, 43%), ulceration (2 out of 7, 29%), and erosion (1 out of 7, 14%) in the esophagus. Serum MPA levels were available in 7 patients, 6 of whom had abnormal duodenal apoptotic counts. On follow-up, available for 16 patients, symptoms improved in all the patients whose dose was decreased or whose medication was withdrawn (10 out of 10) and symptoms persisted in all the patients whose dose was not altered (6 out of 6). Follow-up biopsies after reduction of the medication dose were available for 1 patient and showed substantial reduction in apoptosis. In contrast, follow-up biopsies from 1 patient whose dosage was not altered showed persistent abnormal apoptotic counts in the duodenum. Conclusions As noted by others (Parfitt JR, Jayakumar S, Driman DK. Am J Surg Pathol. 2008; 32:1367–1372), mycophenolate mofetil-associated injury of the upper GI tract, like that in the colon, is characterized by prominent apoptosis similar to that of mild or grade I graft-versus-host-disease injury. We offer apoptotic count guidelines, which we hope will facilitate recognition of mycophenolate mofetil-associated injury in the upper GI tract.

Sevelamer crystals in the gastrointestinal tract (GIT): A new entity associated with mucosal injury

We report the first description of sevelamer crystals (Renagel and Renvela, Genzyme; phosphate-lowering agents) in the gastrointestinal tract. We prospectively collected cases with novel, histologically identical crystals from 4 major academic centers over a 1-year period and studied pertinent clinicopathologic features. Sevelamer usage in the setting of chronic kidney disease was demonstrated in all cases (n=15 total cases, 7 patients). Sites of involvement included the esophagus (n=2), small bowel (n=2), and colon (n=11). The background mucosa was normal in only 1 case. Notable mucosal abnormality included chronic mucosal damage (n=5), acute inflammation (n=4), inflammatory polyp (n=2), extensive ulceration (n=2), ischemia (n=1), and necrosis (n=1). In general, sevelamer crystals displayed broad, curved, and irregularly spaced “fish scales” with a variably eosinophilic to rusty brown color on hematoxylin and eosin (H&E) staining and violet color on periodic acid-Schiff-alcian special staining with diastase (PAS/D). To validate these findings, sevelamer tablets (Renvela) were crushed and submitted for histologic processing; the findings were identical to those in the patient specimens. The possibility of Kayexalate (sodium polystyrene sulfonate) and cholestyramine had been raised in error. However, Kayexalate has narrow, rectangular “fish scales” and is violet on H&E and magenta on PAS/D; cholestyramine lacks internal “fish scales,” is bright orange on H&E, variably gray or hot pink on PAS/D, and is unassociated with mucosal injury. Further study is required to determine whether sevelamer plays a causal role in these injuries; however, its crystal is an important mimic of both Kayexalate and choleystyramine. As the history of sevelamer administration was not documented in any pathology requisition, awareness of sevelamer’s characteristic morphology is crucial to avoid the diagnostic pitfalls of its mimics.

Adenocarcinoma of the small intestine: a multi-institutional study of 197 surgically resected cases

Small intestinal adenocarcinoma is a rare malignant neoplasm, and its clinicopathologic characteristics have not been well elucidated. A total of 197 small intestinal adenocarcinoma cases were collected from 22 institutions in South Korea and were evaluated for clinicopathologic factors that affect the prognosis of small intestinal adenocarcinoma patients using univariate and multivariate analyses. The mean patient age was 59 years, and the male-to-female ratio was 1.7:1. Tumors were located in the duodenum of 108 cases (55%), the jejunum in 59 (30%), and the ileum in 30 (15%). Predisposing conditions were observed in 23 cases (12%), including 17 cases with sporadic adenomas, 3 with Peutz-Jeghers syndrome, 2 with Meckel diverticulum, and 1 with Crohn disease. Synchronous or metachronous malignant tumors were identified in 31 cases (16%), including 13 colorectal and 10 stomach cancers. About 90% of tumors were classified as either pT3 (63 cases) or pT4 (112 cases). The median survival time for all small intestinal adenocarcinoma patients was 39.7 months. Compared with small intestinal adenocarcinomas without accompanying sporadic adenomas, small intestinal adenocarcinomas with accompanying adenomas were more well differentiated (P < .0001), with a more polypoid growth pattern (P < .0001), a lower pT classification (P < .0001), less perineural invasion (P = .01), and less lymphatic invasion (P = .03). Small intestinal adenocarcinoma patients with associated sporadic adenomas (77%) had a significantly better 5-year survival rate than those without sporadic adenomas (38%, P = .02). By univariate analysis, small intestinal adenocarcinoma patients had significantly different survival based on pT classification (P = .003), lymph node metastasis (P < .0001), distal location (jejunal and ileal carcinomas) (P = .003), retroperitoneal tumor seeding (P < .0001), vascular invasion (P = .007), lymphatic invasion (P = .001), peritumoral dysplasia (P = .004), and radiation therapy (P = .006). By multivariate analysis, lymph node metastasis (P = .01) and distal location (P = .003) were independent predictors of a worse prognosis. In conclusion, (1) small intestinal adenocarcinomas are diagnosed at an advanced disease stage; therefore, the development of strategies for detection at an earlier stage is needed. (2) Small intestinal adenocarcinoma patients with an adenomatous component had a better survival than those without an adenomatous component. (3) Lymph node metastasis and distal location (jejunum and ileum) of tumor are the most important independent prognostic factors.

Triheptanoin for glucose transporter type I deficiency (G1D): modulation of human ictogenesis, cerebral metabolic rate, and cognitive indices by a food supplement.

IMPORTANCE Disorders of brain metabolism are multiform in their mechanisms and manifestations, many of which remain insufficiently understood and are thus similarly treated. Glucose transporter type I deficiency (G1D) is commonly associated with seizures and with electrographic spike-waves. The G1D syndrome has long been attributed to energy (ie, adenosine triphosphate synthetic) failure such as that consequent to tricarboxylic acid (TCA) cycle intermediate depletion. Indeed, glucose and other substrates generate TCAs via anaplerosis. However, TCAs are preserved in murine G1D, rendering energy-failure inferences premature and suggesting a different hypothesis, also grounded on our work, that consumption of alternate TCA precursors is stimulated and may be detrimental. Second, common ketogenic diets lead to a therapeutically counterintuitive reduction in blood glucose available to the G1D brain and prove ineffective in one-third of patients. OBJECTIVE To identify the most helpful outcomes for treatment evaluation and to uphold (rather than diminish) blood glucose concentration and stimulate the TCA cycle, including anaplerosis, in G1D using the medium-chain, food-grade triglyceride triheptanoin. DESIGN, SETTING, AND PARTICIPANTS Unsponsored, open-label cases series conducted in an academic setting. Fourteen children and adults with G1D who were not receiving a ketogenic diet were selected on a first-come, first-enrolled basis. INTERVENTION Supplementation of the regular diet with food-grade triheptanoin. MAIN OUTCOMES AND MEASURES First, we show that, regardless of electroencephalographic spike-waves, most seizures are rarely visible, such that perceptions by patients or others are inadequate for treatment evaluation. Thus, we used quantitative electroencephalographic, neuropsychological, blood analytical, and magnetic resonance imaging cerebral metabolic rate measurements. RESULTS One participant (7%) did not manifest spike-waves; however, spike-waves promptly decreased by 70% (P = .001) in the other participants after consumption of triheptanoin. In addition, the neuropsychological performance and cerebral metabolic rate increased in most patients. Eleven patients (78%) had no adverse effects after prolonged use of triheptanoin. Three patients (21%) experienced gastrointestinal symptoms, and 1 (7%) discontinued the use of triheptanoin. CONCLUSIONS AND RELEVANCE Triheptanoin can favorably influence cardinal aspects of neural function in G1D. In addition, our outcome measures constitute an important framework for the evaluation of therapies for encephalopathies associated with impaired intermediary metabolism.

Pdx1 Expression in Pancreatic Precursor Lesions and Neoplasms

Pancreatic and duodenal homeobox (Pdx1) is a homeobox transcription factor required for the embryonic development of the pancreas. Pdx1 expression has been earlier identified in pancreatic ductal adenocarcinomas and endocrine neoplasms. This study characterizes Pdx1 protein expression in pancreatic precursor lesions and neoplasms, including pancreatic intraepithelial neoplasia (PanIN, n=32), intraductal papillary mucinous neoplasm (IPMN, n=88), mucinous cystic neoplasm (MCN, n=3), acinar cell carcinoma (ACC, n=8), pancreatic endocrine neoplasm (PEN, n=44), pancreatoblastoma (PB, n=1), solid pseudopapillary neoplasm (n=8), invasive ductal adenocarcinoma (n=67), and nondysplastic ductal epithelium. A mouse monoclonal antibody for Pdx1 was used to examine archived surgical pathology cases and tissue microarrays containing >655 tissue cores from more than 250 pancreatic specimens. Immunohistochemical labeling for Pdx1 was performed using standard methods and scored for percentage and intensity of nuclear labeling. Among non-neoplastic pancreatic tissues, Pdx1 nuclear protein was expressed in islet cells, cells of the centroacinar cell compartment, and non-neoplastic ductal epithelium. No expression of Pdx1 was seen in non-neoplastic acinar cells. Among pancreatic neoplasms, Pdx1 consistently labeled >50% of the tumor cells in 87.5% of ACC cases and 38.6% of PEN cases. Pdx1 expression was variable in invasive ductal adenocarcinoma and precursor lesions of ductal adenocarcinomas (PanIN, IPMN, and MCN). A single case of PB was examined and it showed Pdx1 in the acinar component, but no expression in squamoid nests. Solid pseudopapillary neoplasms did not express Pdx1. This study shows Pdx1 expression in precursor lesions of ductal adenocarcinomas, PEN, ACC, and a case of PB. In the immunohistochemical evaluation of neoplasms of the pancreas, Pdx1 expression is not a finding specific to PENs and ductal adenocarcinomas, but also occurs in precursor lesions (PanIN, IPMN, MCN) and other neoplasms of the pancreas.

Diagnostic Yield of Clinical Next-Generation Sequencing Panels for Epilepsy

Diagnostic Yield of Clinical Next-Generation Sequencing Panels for Epilepsy During the past 2 years, next-generation DNA sequencing (NGS) has become a widespread diagnostic tool in neurology. Several studies have addressed the diagnostic yield and cost of NGS relative to other types of DNA testing. G-banded karyotyping identifies chromosomal aberrations and has a 3% diagnostic yield for unexplained developmental disabilities or other congenital anomalies.1 In comparison, chromosomal microarrays detect gene copy number variations and have a yield of 15% to 20% for the same disorder categories.1 Next-generation DNA sequencing, in the format of wholeexome sequencing (WES), can be diagnostic in 25% of neurogenetic cases.2 Similarly, whole-genome sequencing (WGS) with NGS has a reported diagnostic yield of 27% in children and adults with a broad variety of diseases.3 In contrast to WES and WGS, targeted NGS panels focus on subsets (dozens to hundreds) of genes associated with specific phenotypes. For example, targeted NGS directed at a single disease category, such as congenital glycosylation disorders, has a reported diagnostic yield of 14.8%.4 Given the prevalence of pediatric epilepsy, we set out to critically assess the diagnostic yield of an NGS panel for epilepsy in a pediatric tertiary care hospital.