Next Generation Children. Association for Clinical Genetic Science meeting 27th June 2017 Alba Sanchis Juan,

Transcription

1 Next Generation Children Association for Clinical Genetic Science meeting 27th June 2017 Alba Sanchis Juan,

2 NGC project Genetic testing for patients from NICU and PICU with a suspected genetic disorder Costly, time-consuming, and inconclusive Diagnostic odyssey Trio analysis of WGS data Feasibility of the implementation of WGS in routine practice Potential of a rapid turnaround service 2 weeks 2

3 NGC project Why WGS? SV, CNV and GC rich content regions Mean coverage 30x Why trios? Fast and accurate analysis of de novo variants Likely to be enriched Sample size 400 samples planned (~125 samples so far) 85% families are trios 3

4 Workflow Clinician Validation Family recruitment Decision Sample processing Data interpretation Data generation 4

5 Workflow NICU and PICU Clinician Cambridge NHS Genetics Laboratory Validation Family recruitment NGC Recruitment team Multidisciplinary team Decision Sample processing Cambridge NHS Genetics Laboratory Research analyst and bioinformatics team Data interpretation Data generation Illumina, UK 5

6 Workflow NICU and PICU Clinician Cambridge NHS Genetics Laboratory Validation Family recruitment NGC Recruitment team Multidisciplinary team Decision 2 days 3 days 10 days Sample processing Cambridge NHS Genetics Laboratory Research analyst and bioinformatics team Data interpretation Data generation Illumina, UK 6

7 Workflow Clinician Validation Family recruitment Decision Sample processing Data interpretation Data generation 7

8 Family recruitment NICU and PICU screening Counseling + consents Phenotyping Blood collection 8

9 Family recruitment NICU and PICU screening Genetic cause of disease is suspected Counseling + consents Phenotyping Blood collection 9

10 Family recruitment NICU and PICU screening Counselling + consents Brief and long participant consent Phenotyping Blood collection 10

11 Family recruitment NICU and PICU screening Counseling + consents Phenotyping Phenotypes recorded as HPO Blood collection 11

12 Family recruitment NICU and PICU screening Counseling + consents Phenotyping Blood collection 12

13 Family recruitment NICU and PICU screening Counseling + consents Phenotyping High quality and concentration of DNA from low amount of blood from premature infants Short timeframe Blood collection 13

14 Workflow Clinician Validation Family recruitment Decision Sample processing Data interpretation Data generation 14

15 Data generation DNA Sequencing Computing Improved WGS pipeline from NIHR Bioresource Rare Diseases, in collaboration with Illumina, UK research pipeline Challenges of setting up the pipeline Pipeline validation 2 different pipelines Array SV validation 15

16 Data generation SV CNV Annotated variants Candidate variant/s Annotation Filtering SNV InDels Filtering criteria 1. Quality of the variant 2. Allele Frequency 3. Functional coding impact OR known pathogenic variant in HGMD Pro or ClinVar 4. Segregation (de novo, biallelic, X-linked, mitochondrial) 5. Curated gene list + HPO 16

17 Workflow Clinician Validation Family recruitment Decision Sample processing Data interpretation Data generation 17

18 Data interpretation ACMG standards and guidelines Candidate variant/s Known gene Quality of the variant Phenotype and inheritance of the gene Pathogenic variant/s 18

19 Workflow Clinician Validation Family recruitment Decision Sample processing Data interpretation Data generation 19

20 Decision, validation and report Research MDT meeting Discussion of the variants selected by the analyst Clinical decision Variant validation Accredited validation in Cambridge NHS diagnostic laboratory Report to clinician Only pathogenic and likely pathogenic variants are reported 20

21 Results 29 trios analysed 3 potential diagnoses to date 21

22 Case 1 Clinical history Male NICU Premature (32 weeks) Respiratory distress at birth, ventilated 26 days Severe congenital hypotonia and myopathy Lower limb contractures (knee and feet) Died after planned extubation and transfer to hospice within 24 hours 22

23 Case 1 Investigations ECG and echocardiogram EEG, MRI brain Muscle biopsy Microarray, Prader Willi, SMA and myotonic dystrophy all negative 23

24 Case 1 Variant Gene Gene phenotype Effect Evidence XLR missense variant NM_ c.1373t>c MTM1 Myotubular myopathy, XLR Likely pathogenic (V) PM1, PM2, PP2, PP3 24

25 Case 1 MTM1 X-linked recessive myotubular myopathy gene Rare condition Severe congenital hypotonia due to a myopathy Respiratory failure Early death 25

26 Case 1 Hemizygous MTM1 Proband Mother Heterozygous MTM1 Father 26

27 Case 2 Clinical history Female PICU, aged 13 years Presented with a large pericardial effusion and fever No structural heart abnormalities Considered underlying lymphoma Developmental delay Dysmorphic features suggestive of Noonan's syndrome 27

28 Case 2 Investigations Microarray: no clinically significant abnormalities Gene panel sequencing, including Noonan syndrome and Costello syndrome 28

29 Case 2 Variant Gene Gene phenotype Effect Evidence denovo missense variant NM_ c.1513c>t BRAF Noonan syndrome, AD Pathogenic (IIIb) PM1, PS2, PM2, PP2, PP3, PP5 29

30 Case 2 Heterozygous de novo BRAF Proband Mother Father 30

31 Case 3 Clinical history Female NICU Term delivery (41 weeks) Emergency caesarian section due to fetal distress Birth weight and head circumference normal, Apgar scores low Hypoxic-Ischemic Encephalopathy (HIE) Grade 2 Neonatal cooled to 33.5 o C core temp for 3 days No seizures but abnormal EEG Discharged well at day 9 31

32 Case 3 Investigations No routine microarray or specific genetic testing performed as not indicated Recruited due to an unusual presentation of HIE 32

33 Case 3 Complex de novo structural variant called from NGS 2 duplications on chromx Kb Inversion of 458 Kb, that disrupts CDKL5 33

34 Disruption of CDKL5 34

35 Disruption of CDKL5 Duplication Duplication Inversion 35

36 Disruption of CDKL5 36

37 Disruption of CDKL5 37

38 Case 3 CDKL5 Haploinsufficiency of CDKL5 causes EIEE in females Associated with epilepsy and poor long term developmental profile Cautious interpretation needed Illustrates the power of WGS 38

39 Summary We have established a pipeline for the rapid turnaround of WGS data in patients from NICU and PICU Early diagnosis can have a significant impact on potential terminal care decisions and timely reproductive decision making Children in NICU/PICU may have multiple etiologies and/or complex phenotypes where WGS trio analysis provides a significant contribution to early diagnosis and management of known diseases Early days 39

40 Acknowledgments Department of Paediatrics Amanda Ogilvy-Stuart Gusztav Belteki Marion Bohatschek Cristine Costa Samantha O Hare Wilf Kelsall Angela D Amore Shazia Hoodbhoy Stergios Papakostas Rajiv Chaudhary Yogen Singh Kathryn Beardsall Rosalie Campbell Roddy O'Donnell Nazima Pathan Shruti Agrawal Riaz Kayani Susan Broster Cambridge Diagnostic Laboratory Isabelle Delon Joanne Harley Natalie Holder Steve Abbs Illumina Sean Humphray, Claire Allen Joseph Hughes John Peden Cambridge BRC Departments of Medical Genetics and Paediatrics Lucy Raymond flr24@cam.ac.uk David Rowitch Topun Austin Ricardo Branco Kelly Spike Audrienne Sammut Hilary Wong Helen Dolling Eleanor Dewhurst Keren Carss Courtney French Kathy Stirrups Karyn Mégy Chris Penkett Olga Shamardina Salih Tuna Sri Deevi Willem Ouwehand 40

41 41

42 Proband Father Mother 42

43 Case3 Also duplication of RAI2 and SCML2 Not well described genes 43