ORIGINAL RESEARCH published: 03 August 2015 doi: 10.3389/fped.2015.00067 Clinical impact and cost-effectiveness of whole exome sequencing as a diagnostic tool: a pediatric center’s experience C. Alexander Valencia 1*, Ammar Husami 1, Jennifer Holle 1, Judith A. Johnson 1, Yaping Qian 2, Abhinav Mathur 1, Chao Wei 1, Subba Rao Indugula 1, Fanggeng Zou 1, Edited by: Haiying Meng 1, Lijun Wang 1, Xia Li 1, Rachel Fisher 1, Tony Tan 1, Amber Hogart Begtrup 1, M. Z. A. Bhuiyan, Kathleen Collins 1, Katie A. Wusik 1, Derek Neilson 1, Thomas Burrow 1, Elizabeth Schorry 1, University Hospital Lausanne, Robert Hopkin 1, Mehdi Keddache 1, John Barker Harley 3,4, Kenneth M. Kaufman 3,4 and Switzerland Kejian Zhang 1 Reviewed by: Peter Turnpenny, 1 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Royal Devon and Exeter NHS Cincinnati College of Medicine, Cincinnati, OH, USA, 2 Myriad Genetics Laboratories, Inc., Salt Lake City, UT, USA, 3 Center for Foundation Trust, UK Autoimmune Genomics and Etiology (CAGE), Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, Alex Vincent Postma, University of Cincinnati College of Medicine, Cincinnati, OH, USA, 4 US Department of Veterans Affairs Medical Center, Academic Medical Center, Cincinnati, OH, USA Netherlands Xusheng Wang, St. Jude Children’s Research Background: There are limited reports of the use of whole exome sequencing (WES) Hospital, USA as a clinical diagnostic tool. Moreover, there are no reports addressing the cost burden *Correspondence: associated with genetic tests performed prior to WES. C. Alexander Valencia, Division of Human Genetics, Objective: We demonstrate the performance characteristics of WES in a pediatric Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, setting by describing our patient cohort, calculating the diagnostic yield, and detailing the MLC7016, Cincinnati, OH 45229, USA patients for whom clinical management was altered. Moreover, we examined the potential [email protected] cost-effectiveness of WES by examining the cost burden of diagnostic workups. Specialty section: Methods: To determine the clinical utility of our hospital’s clinical WES, we performed This article was submitted to Genetic Disorders, a section of the journal a retrospective review of the first 40 cases. We utilized dual bioinformatics analyses Frontiers in Pediatrics pipelines based on commercially available software and in-house tools. Received: 13 May 2015 Accepted: 13 July 2015 Results: Of the first 40 clinical cases, we identified genetic defects in 12 (30%) patients, Published: 03 August 2015 of which 47% of the mutations were previously unreported in the literature. Among the Citation: 12 patients with positive findings, seven have autosomal dominant disease and five have Valencia CA, Husami A, Holle J, autosomal recessive disease. Ninety percent of the cohort opted to receive secondary Johnson JA, Qian Y, Mathur A, Wei C, Indugula SR, Zou F, Meng H, findings and of those, secondary medical actionable results were returned in three cases. Wang L, Li X, Fisher R, Tan T, Among these positive cases, there are a number of novel mutations that are being Hogart Begtrup A, Collins K, Wusik KA, Neilson D, Burrow T, reported here. The diagnostic workup included a significant number of genetic tests Schorry E, Hopkin R, Keddache M, with microarray and single-gene sequencing being the most popular tests. Significantly, Harley JB, Kaufman KM and genetic diagnosis from WES led to altered patient medical management in positive cases. Zhang K (2015) Clinical impact and cost-effectiveness of whole exome sequencing as a diagnostic tool: a pediatric center’s experience. Abbreviations: ACMG, American College of Medical Genetics; CAP, College of American Pathologists; CASAVA, consensus Front. Pediatr. 3:67. assessment of sequence and variation; IRB, Institutional Review Board; MODY, maturity onset diabetes of the young; VUCS, doi: 10.3389/fped.2015.00067 variant of unknown clinical significance; WES, whole exome sequencing. Frontiers in Pediatrics | www.frontiersin.org 1 August 2015 | Volume 3 | Article 67 Valencia et al. Utility of clinical exome sequencing Conclusion: We demonstrate the clinical utility of WES by establishing the clinical diagnostic rate and its impact on medical management in a large pediatric center. The cost-effectiveness of WES was demonstrated by ending the diagnostic odyssey in positive cases. Also, in some cases it may be most cost-effective to directly perform WES. WES provides a unique glimpse into the complexity of genetic disorders. Keywords: whole exome sequencing, next generation sequencing, diagnosis, children, clinical utility, pediatrics Introduction Patients, Materials, and Methods Mendelian diseases account for a significant number of pedi- Clinical Samples atric disorders. Recently, a systematic review of the records of Forty pediatric patients referred by medical specialists (Medical 5,747 consecutive admissions in 1996 to Rainbow Babies and geneticists 77%, Immunologists 15%, Cardiologists 3%, and others Children’s Hospital (Cleveland, OH, USA) found an underly- 3%) for exome sequencing have had the analysis and results dis- ing disorder with a significant genetic component in 71% of closure completed. The patients in this cohort had diverse clinical admitted children (1). Diagnosing patients with complex phe- features and these are summarized in Table 1. Before referral, notypes generally involves physical examination, detailed family all patients had undergone extensive diagnostic evaluations (e.g., history, complementary tests such as radiography and metabo- aCGH microarray, targeted gene tests/panels, metabolic screen- lite analysis, and genetic testing. A significant proportion of ing, clinical genetic evaluations, and other laboratory workup) patients undergo extensive genetic testing including karyotyp- that did not lead to a unifying diagnosis. Consent for clinical ing, array-based comparative genomic hybridization, Sanger WES was obtained from the patients and/or their family. Internal sequencing, and multigene next-generation sequencing panels review board (IRB) approval was obtained at Cincinnati Children’s but still remain undiagnosed (2). Accurate diagnosis poten- Hospital Medical Center (CCHMC) for this retrospective study. tially benefits patients and their families by altering clinical management, predicting recurrence risks, providing progno- Whole Exome Sequencing and Sanger sis, and ending the diagnostic odyssey that is invasive, time consuming and costly. Clinical diagnosis is therapeutic in its Confirmation own right for the patient/family, as a result of ending the WES and analysis protocols were developed and validated by “diagnostic odyssey” – quite apart from the tangible clinical the CCHMC molecular genetics laboratory of the Division of benefits. Human Genetics. Briefly, genomic DNA samples from patients The advent of next-generation sequencing technologies has were fragmented by sonication, ligated to Illumina multiplexing provided an opportunity to affordably screen a patient’s entire paired-end adapters, amplified by means of a polymerase-chain exome to establish genetic basis of disease (3–9). The “exome” reaction and hybridized to biotin-labeled NimbleGen V3 exome is the component of the genome that predominantly encodes capture reagent (Roche NimbleGen). Hybridization was achieved at 47°C for 64–72 h. After washing and reamplification, paired- protein; these segments are referred to as “exons” and can × include non-coding exons. The exome comprises about 1% of end sequencing (2 100 bp) was performed on the Illumina the genome and is, so far, the component most likely to include HiSeq 2500 platform to provide average sequence coverage of more than 100×, with more than 97% of the target bases having at interpretable mutations that result in clinical phenotypes. Whole × exome sequencing (WES) involves determination of the DNA least 10 coverage. Clinically relevant variants, from proband and sequence of most of these protein-encoding exons and may parental samples (whenever available), were confirmed by Sanger include some DNA regions that encode RNA molecules that are sequencing. not involved in protein synthesis. The utility of WES to iden- tify variants causative of Mendelian disorders has been clearly Data Analysis and Annotation demonstrated in identifying novel candidate genes for Miller syn- To aid in the clinical interpretation of variants, data were anno- drome, Fowler syndrome, Perrault Syndrome, and many other tated and analyzed using two pipelines: (1) next gene pipeline disorders (10–17). However, the clinical utility of exome sequenc- (NGP) and the (2) genome analysis toolkit (GATK)/golden helix ing in pediatric patients needs further examination (18–22). pipeline (GGHP). For NPG, output data from the Illumina HiSeq Herein, we report a series of our first 40 consecutive pedi- 2500 were converted from bcl files to FastQ files using the Illumina atric cases that were referred for WES in a clinical laboratory. consensus assessment of sequence and variation (CASAVA) soft- We demonstrate the clinical utility of WES in a pediatric set- ware, version 1.8, and mapped to the reference haploid human- ting by describing our patient cohort, calculating the diagnos- genome sequence (hg19) with NextGene 2.3.4 using default set- tic yield, detailing the cases in which clinical management was tings. Passing quality