Integration of Next-Generation Sequencing into Epilepsy Clinical Care. Michelle Demos University of British Columbia BC Children s Hospital

Transcription

1 Integration of Next-Generation Sequencing into Epilepsy Clinical Care Michelle Demos University of British Columbia BC Children s Hospital

2 No Disclosures

3 Learning Objectives To review: the impact of using next-generation sequencing (NGS) in epilepsy its integration into clinical practice

4 Case June week old female infant: Presented Day 3 with focal motor seizures: Head/eye right deviation, extension all limbs, and apnea ongoing seizures despite phenobarbital, levetiracetam, topiramate, and midazolam infusion; intubated and ventilated 3 days No risk factors for acquired brain injury Family history unremarkable Mild hypotonia on exam 4

5 Guella I et al Neurology Genetics 2016 Case Day 8

6 Guella I et al Neurology Genetics 2016 Case Day 8

7 Guella I et al Neurology Genetics 2016 Case Day 8

8 Case - Normal Investigations Head MRI Chromosome microarray CSF studies (including neurotransmitters and infection) Transferrin isoforms Plasma amino acids Acylcarnitine profile Ceruloplasmin and copper Ammonia Lactate Urine organic acids Urine purine and pyrimidines Urine AASA Homocysteine

9 Case Diagnosis Focal motor seizures-neonatal onset Etiology: Unknown likely Genetic Diagnostic Approach and management?

10 Outline Epilepsy and Next-generation sequencing why? Next-generation sequencing as a diagnostic tool in the epilepsy clinic who, when, what?

11 Outline Epilepsy and Next-generation sequencing why? Background Discovery and Diagnostic Yield Mosaicism Economic and Clinical Impact BC Study

12 Causes of epilepsy Genomic imbalances Mitochondrial Ref. Thomas, R. H. & Berkovic, S. F. Nat.Rev.Neurol. 2014

13 Sequence analysis Then Sanger sequencing PCR fragments > sequence > analyze gold standard sequence quality, low throughput First human genome: 10 years, 3 billion

14 Next-generation sequencing Now Massively parallel sequencing Sequence millions of fragments simultaneously Whole genome, exome (~1% genome), gene panel Patient s clinical genome: $9000 ($ trio) in ~16 weeks Partners in Health, Harvard 2017.

15 Exome contains 85% of disease-causing mutations

16 Limitations: Next-generation sequencing WES and panels Coding regions base pairs flanking exonic sequence are typically covered Coverage Repeat sequence(s) within exonic sequence Copy number variants Abnormal methylation Mosaicism deep sequencing Genomics Education Programme.

17 ALG13 DEPDC5 SYNGAP1 CHD2 KCNT1 Myers CT, Mefford HC Genome Medicine 2015.

18 Impact: Discovery Discovery: ~ 600 known associated epilepsy genes and more than 250 candidate genes

19 Diagnostic Yield Number of patients Patient type Method Yield 349 Treatment resistant and early onset (< 1 yr) 216 Neonatal-adult onset Benign-severe Targeted 30 genes; 90 genes 20.3% Targeted 46 genes 23% Highest early onset and EE 6 Severe early onset WGS 67%* 293 Epilepsy plus WES 38.2%* Highest in early onset EE years MRI negative focal epilepsy + family history Parrini E et al 2016; Moller RS et al 2016; Martin HC et al 2014; Helbig KL et al Genet Med 2016; Perucca P et al Targeted WES (64 genes) 12.5% Highest in earlier onset

20 From: Early-Life Epilepsies and the Emerging Role of Genetic Testing JAMA Pediatr. 2017;171(9): doi: /jamapediatrics Berg AT et al. Figure Legend: Genetic Test Yields in Children With Otherwise Unexplained EtiologyGenetic test results in children with unexplained etiology. Vertical bars indicate 95% CIs. Copyright 2017 American Medical Association. All Rights Reserved.

21 What have we learned? Patients with a genetic diagnosis: Early onset epilepsy, treatment resistant, epileptic encephalopathy, and/or presence of additional neurological condition (eg Global developmental delay/ intellectual disability) Low yield in typical benign electroclinical syndromes De novo dominant variants (Epileptic encephalopathies) Phenotypic and genotypic heterogeneity broader phenotype; atypical presentations

22 Genotypic Heterogeneity: Dravet syndrome and its mimics: Beyond SCN1A Epilepsia 7 SEP 2017 DOI: /epi

23 ILAE Helbig I, Krause R Phenotypic Heterogeneity Genotype-Phenotype correlation

24 Somatic Mutations causing epilepsy Poduri et al Science 2013 LIS1 AKT3

25 Focal cortical dysplasia (II) may also reflect somatic mutations mtor TSC1 FCD is one of the most common causes of treatment resistant focal epilepsy somatic mutations in mtor (15%); TSC1/2 (12% in mtor negative); rare PIK3CA Hyperactivation mtor kinase therapeutic implications Lim JS et al Nat. Med 2015; Nakashima M et al Ann Neurol 2015; Moller RS et al Neurology Genetics 2016; Lim JS et al Am J Hum Genet 2017.

26 epilepsy probands with pathogenic/likely pathogenic variants in CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A or SCN2A identified via epilepsy panel or WES 9.5% mosaic probands 3.7% mosaic parent 73% asymptomatic Enhanced detection of mosaicism can provide: definitive molecular diagnosis to patients who may have received negative results from less sensitive testing More accurate estimates of recurrence risk (33% vs negligible)

27 Cost-effectiveness 2016 Economic Impact? WES identified novel mutations in known epilepsy genes in all 4 patients studied Analyzed cost savings that could be accrued if WES was performed earlier in the diagnostic odyssey Average of traditional tests until WES performed Time spend in the diagnostic odyssey 1.4 to 8.2 years Total cost of traditional tests ranged from $9,015 to $35,483 WES trio cost $6,100; turnaround time 12 weeks This study and others supporting its use as first-tier diagnostic test especially in early-onset disorders

28 Next-Generation Diagnostics in Epilepsy Benefits: Diagnostic yield, Clinical Impact, (Gene Discovery) Cost savings; time to diagnosis Challenges: Availability, limitations, analysis, variants of unclear significance, and incidental findings

29 Next-Generation Diagnostics in Epilepsy: Potential Negative Clinical Impact Identifying a disease without specific treatment Inaccurate interpretation of a variant Causal variant not identified by sequencing Discrimination regarding life, disability or long-term care insurance Incidental findings Genetic counselling before testing

30 Clinical Impact of a Genetic Diagnosis Earlier diagnosis including disorders with specific treatment implications medication decisions eg B6 dependent epilepsy specific treatments eg ketogenic diet in GLUT1 surgical decisions eg SCN1A informs research for targeted therapy precision medicine More accurate counselling regarding prognosis and recurrence risk; prenatal testing Prevent additional unnecessary investigations Family support groups

31 BC Epilepsy Genomics Study Djavad Mowafaghian Centre for Brain Health at UBC Hospital

32 Overall Objective Generate data that will demonstrate to the BC Ministry of Health the value of using Whole Exome Sequencing (WES) in the clinical care of children with unexplained early onset epilepsy Diagnostic Yield Time to diagnosis Cost Treatment Impact 32

33 Epilepsy & Genomics Study: Subjects and Methods Epilepsy onset age 5 and under cause of epilepsy is unknown based on clinical evaluation (head MRI, CMA and EEG) 160 eligible participants group 1/prospective: 40 individuals with epilepsy < 6 months group 2/retrospective: 120 individuals with epilepsy > 6 months (standard clinical approach to genetic testing) Targeted analysis of WES (565 genes): Results available: 151 (41 Prospective; 110 Retrospective)

34 Diagnostic Yield and Treatment Implications Diagnostic Yield Treatment Implications 46% Definite/Likely 25% Possible 10% No Diagnosis 65%

35 Inheritance Diagnostic Yield Xlinked de novo 19% Xlinked 6% Recessive 8% Dominant 8% Dominant de novo 59%

36 Definite/Likely Possible KCNQ2 x 3 GABRA1 x 2 KCNQ2 x 3 STXPB1 x 2 KCNT1 PIGA SCN1A x 7 SCN8A KCNT1 SMC1A ARX GABRB3 x 2 POLG ATP1A3 ARHGEF9 ADSL DYNC1H1 PCDH19 MED23 CNKSR2 SCN3A ATP1A2 DYRK1A x 2 CHD2 ALG13 MECP2 x 2 SCN1B PAFAH1B1 DCX CACNA1H CDKL5 COL4A2 TUBB2B HNRNPU SLC35A2 SLC1A2 YWHAG Guella I et al De-novo mutations in YWHAG cause early-onset epilepsy. Am J Hum Genet August 2017.

37 Clinical Features in Patients with and without a Genetic Diagnosis Patients* All (n=151) Age epilepsy onset mean (range) 21 months (.2 60) Males Epileptic Encephalopathy Treatment DD/ID Autism MRI Resistant 1 Abnormal 42% 50% 86% 77% 21% 34% Definite or Likely Diagnosis (n=37) No Diagnosis (n=99) 14 months (.3 52) 23 months (0.2 60) 38% 59% 84% 86% 24% 38% 43% 47% 87% 73% 20% 30% *We excluded individuals with variants of unknown significance or possible genetic diagnosis. 1 Treatment resistant refers to failure to respond to 2 or more appropriate anti-seizure medications. DD/ID=Developmental Delay/Intellectual Disability.

38 Using data on first 50 patients Prospective Cohort Epilepsy onset Genetic diagnosis = 3.6 months Epilepsy Onset Diagnosis Study Enrolment 71 days 38 days Retrospective Cohort Epilepsy onset Genetic diagnosis = 7.6 years Epilepsy Onset Study Enrolment 2711 days 54 days Diagnosis

39 Cost estimation Patient diagnostic tests (biochemical, imaging, genetic, electrophysiology, biopsies) were obtained from medical charts and electronic records Costs estimated from multiple sources and academic and/or hospital pricing was used Inpatient hospitalization costs, outpatient visits such as clinic visits, and indirect costs such as parental time off work for medical visits related to their child s epilepsy were not included

40 Cost estimation The mean total cost related to the diagnosis of epilepsy was: $4,524 (range $1,223-$7,852) for the prospective cohort and $8,344 (range $3,319-$17,579) for the retrospective cohort Alternative scenario for diagnostic testing: $3,234/patient MRI ($686), EEG ($188), chromosome microarray (CMA) ($860) and WES testing with Sanger sequencing validation ($1500) The potential average savings of targeted WES in the diagnostic workup constitute $1,290 per prospective patient and $5,110 per retrospective patient.

41 Case Diagnosis Focal motor seizures-neonatal onset Etiology: Unknown likely Genetic Diagnostic Approach and management?

42 Case BC Epilepsy Genomics Study Exome sequencing results 9 months: Heterozygous de novo FGF12 variant c.g155a (p.r52h) Likely Pathogenic

43 Case: 2 weeks to 2 years Seizures responded to phenytoin on Day seizures per month on phenytoin and topiramate Seizure free since 5 months of age on carbamazepine Normal development, examination and head circumference 43

44 Case Significance same FGF12 variant described in at least 6 other affected individuals with severe early onset epilepsy and developmental delay 2 early deaths with cerebellar atrophy FGF12 interacts with sodium channels and gain of function mutation is predicted to increase neuronal excitability Limited information on patients support positive response to sodium channel blocker like phenytoin all started later than our patient, which possibly contributed to their poor outcomes

45 Examples from Treatment Implication Group Phenotype Gene Impact on Management Dravet Syndrome (001) SCN1A Antiepileptic medication change with reduction in episodes of status epilepticus Alpers Syndrome (033) POLG Stopped valproic acid, early palliative care with Canuck Place involvement; prenatal testing Self-limited Familial Neonatal Seizures (104) KCNQ2 Stopped phenobarbital at 2 m; avoided MRI with anesthetic; seizure free and normal at 6 months Hemiplegic migraine plus ATP1A2 Started flunarizine; stopped other antiepileptic medications; significant reduction in seizures and hemiplegic episodes KCNQ2 Encephalopathy (040) KCNQ2 Phenytoin changed to carbamazepine: seizures less frequent and shorter 9 months later. Congenital Disorder Glycosylation IIm (065) SLC35A2 Galactose trial: 6 months later more alert and interactive; no change in seizure frequency or development level. Wilbur C et al Pediatr Neurol 2017.

46 Proband-parent trio-based WES analyses was performed for 23 patients with no immediate genetic diagnosis. A likely/definitive diagnosis was identified in 2 patients: de novo p.ser737tyr SMARCA2 variant de novo p.arg52his FGF12 variant Prioritization criteria: 1. Epileptic encephalopathy A possible diagnosis 2. Onset was identified of seizure in<1y 2 additional patients: Maternally inherited (X-linked) p.arg41trp PIGA variant De novo p.pro369arg KCNQ5 variant collaborative discovery In 11 additional patients we identified a candidate gene supporting evidence is pending. Guella I et al De novo FGF12 mutation in 2 patients with neonatal-onset epilepsy. Neurol Genet Lehman A et al Loss-of-function and gain-offunction mutations in KCNQ5 cause intellectual disability or epileptic encephalopathy. Am J Hum Genet 2017.

47 Outcome Preliminary results from our study helped support the recent BC Ministry of Health approval of sequencing for seizure disorders (SSD) at Neurocode Laboratory Inc Targeted WES Testing applies to infants and children with seizure disorders of unknown etiology referral by neurologists

48 Outline Epilepsy and Next-generation sequencing why? Next-generation sequencing as a diagnostic tool in the epilepsy clinic who, when, what?

49 Who? Unexplained epilepsy, likely genetic (single gene): childhood onset-priority early onset-neonatal/infantile Epileptic encephalopathy Treatment resistant epilepsy Seizures associated with fever (excluding simple febrile seizures) Neurodegenerative disorder/poor prognosis Clinically suggestive inborn error of metabolism Malformation of cortical development Additional neurological features: GDD/ID, paroxysmal neurological features, autism, congenital anomalies Familial epilepsy (at least 2 first-degree) Adapted by Genetic Testing Advisory Committee Recommendation to the Ministry of Health and Long-Term care regarding Criteria for Genetic Testing Related to Epilepsy for Ontario Laboratories October 2016.

50 Not recommended Recognizable typical electroclinical or epilepsy syndrome Typical childhood absence epilepsy Typical Juvenile myoclonic epilepsy Benign Childhood Epilepsy with Centrotemporal Spikes Acquired Epilepsy

51 When? Prerequisites: Epileptologist/Neurologist phenotype EEG, MRI, Chromosome microarray Genetic counselling (before/after) Other testing to consider inborn errors of metabolism not required before testing Early in the diagnostic work-up

52 Gene panel, exome or whole genome? Method Benefits Challenges Recommendations Single gene gold standard No incidentals Cost $720 MECP2 1 Low level mosaicism Low-throughput Heterogeneity Limited indications Clear diagnosis with low heterogeneity Eg Glut-1 (SLC2A1) Targeted panel Coverage More genes No incidentals Cost $950-Rett/Angelman 1 Limited genes tested; misses new genes Diagnosis clear Gene panel depends on degree of heterogeneity WES (targeted analysis) Genes tested new genes Discovery Cost decreasing $ Coverage Incidentals (less if targeted analysis) Diagnosis unclear Heterogeneity high or unknown WGS Coding & Non-coding regions discovery CNVs Cost will likely decrease Data interpretation Incidentals Cost $ mosaicism Near Future? 1 GeneDx Partners in Health 2017.

53 What about my patient? Near future Patient Clear Diagnosis (0 to minimal heterogeneity) Diagnosis unclear or heterogeneity high or unknown Clear Diagnosis (low to high heterogeneity) Single gene request eg CSTB Focused gene panel eg Angelman-like syndrome Comprehensive panel if high heterogeneity Whole genome sequencing Comprehensive panel or targeted WES or WES

54 Summary and Future Directions Advances in Next Generation Sequencing has led to a rapid increase in our understanding of epilepsy genetics, and studies support its use early on in the diagnostic work-up of specific epilepsy cohorts A better understanding of the genetic variation is starting to impact on clinical management precision medicine. Further studies are required to measure its impact on clinical outcome. Sharing of research data and ongoing collaborations will be important in order to continue to identify causative variants and advance targeted therapy to help improve outcome