Validating De novo Mutations Associated with Epileptic Encephalopathy through Sanger Sequencing Ariana Cruz1, Alison M. Muir2, Amy LaCroix2, Gemma L. Carvill2, Heather C. Mefford2 1UW GenOM Project; 2Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, Washington

Introduction Sanger Sequencing Conclusion • Epileptic encephalopathy (EE): a classification of • From the total of 14 variants identified: epilepsy characterized by severe seizures with onset during infancy or childhood with developmental delay and regression Validated 3 • Many cases stem from de novo mutations: mutations De novo 1 present only in the child, not the mother or father False Positives 4

Size exclusion PCR Acquired Require Further Investigation 7 Unaffected mother Unaffected father of DNA Amplification fragments Sequence • Results confirm that validation by Sanger sequencing is Results an important step to ensure that the mutation Affected child identified by next-generation sequencing is truly • Currently intractable to antiepileptic drugs WDR45 c.C611T Missense Mutation present Single nucleotide change results in a • Through targeted next-generation sequencing, a codon for a different amino acid • WDR45 c.C611T is now considered a solved mutation. cohort clinically diagnosed with EE, was screened for U C A A C G G A G U U U A A C The other validated mutations remain unknown mutations within genes known to be causative of EE because inheritance is indefinite Ser Thr Glu Phe Asp • Sanger sequencing was used to: . Determine if the mutation was de novo and C C G • The data obtained will be used to enhance current therefore likely to be pathogenic knowledge of EE and to further the development of . Ensure the mutation is not a false-positive Ser Pro Glu Phe Asp potential treatment Mutation Validations Methods Sample hg19 Amino Acknowledgments Mutation Gene Mutation Validation Inheritance Name Coordinates Acids • My Principal Investigator Dr. Heather C. Mefford e4776-5 c.A1141G 2:166170236 SCN2A missense MET,VAL False + - • My mentor Alison M. Muir • The Mefford Lab (Amy LaCroix, Gemma L. Carvill, Candace Myers, Michele Mehaffey, John Nguyen, Aaron Rosen, Zoe e3927-1 c.C4908G 2:166245224 SCN2A missense ILE,MET Validated No seq Primer Design Thuesmunn, and Nancy Nguyen) e4919-1 c.A969T 5:139494735 PURA stop-lost stop,CYS False + - • Lisa Peterson, Gregory Diggs-Yang, Jonathan Yee, Christopher Moreno, and contributors of GenOM ALVA • Donations from Dr. Anne Dinning and Dr. Michael Wolf e4738-1 c.A1353C 5:161569293 GABRG2 missense LYS,THR No seq No seq • Funding: University of Washington GenOM Project (NIH 5R25HG007153-04) PCR e4687-4 c.A1265G 12:111747851 CUX2 missense ASP,GLY No seq No seq T24802-2 c.G1506T 15:25615824 UBE3A stop-gained TYR,stop False + - References 8058395_1 c.G560A 19:35524755 SCN1B intron,missense none Validated No seq

Gel Electrophoresis e4914-1 c.G1210A 19:50365117 PNKP missense ARG,CYS No seq No seq • Khan, S., & Raidah Al Baradie, R. A., “Epileptic Encephalopathies: An Overview,” Epilepsy Research and Treatment, vol. 2012, e4914-1 c.T1183A 19:50365306 PNKP missense ASN,TYR No seq No seq Article ID 403592, 8 pages, 2012.

e4902-1 c.G3743A 22:32272189 DEPDC5 stop-gained TRP,stop False + -

Sanger Sequencing e4693-1 c.C741G 1:43395390 SLC2A1 missense GLU,ASP No seq No seq

e4699-1 c.T2672C 2:166201174 SCN2A missense ILE,THR No seq No seq Sequence Analysis T20627 c.457delACTG 9:130422380 STXBP1 frameshift none No seq No seq e4695-1 c.C611T X:48933318 WDR45 missense GLY,ASP Validated de novo

University of Washington GenOM Project ALVA 2016