NGS in Diagnostics: a practical example in hereditary cardiomyopathies

Transcription

1 NGS in Diagnostics: a practical example in hereditary cardiomyopathies Patricia Norambuena University Hospital Motol, Prague. 2 nd Faculty of Medicine, Charles University VEP Course - November 6 th - 8th, 2017

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3 Thick Rigid Enlarged Sometimes the heart goes wrong Heart rhythm problems (arrhythmias / channelopathies) Heart structural problems (cardiomyopathies) From Loeys, 2016

4 Cardiomyopathy - heart muscle disease Deterioration of the ability of the myocardium (the heart muscle) to contract, usually leading to heart failure. Typically inherited in an Autosomal Dominant (AD) pattern

5 Hypertrophic Cardiomyopathy Hypertrophy of the left ventricle (LV) >14 mm in absence of any other cardiac or systemic disease that may lead to hypertrophy Myofibrils lose their proper parallel orientation and become disorganized into a pattern known as disarray. Might lead to arrhthymias and Sudden Cardiac Death (SCD) The flow through the heart might be limited Molecular mechanism of the disease is still unclear

6 Hypertrophic Cardiomyopathy Private mutations Population frequency: 1/500 Hypertrophic cardiomyopathy is associated with mutations in sarcomere genes Presents an age-related penetrance Highly heterogenic phenotype From asymptomatic patients to Sudden Cardiac Death (SCD)

7 Hypertrophic Cardiomyopathy In about 50 % of HCM patients, the disease is caused by mutations in the genes coding for cardiac sarcomere proteins. Most commonly affected are the MYBPC3 and MYH7 genes.

8 Inherited cardiac conditions may lead to Sudden Cardiac Death (SCD)

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11 Sudden Cardiac Death PREVENTION Affected individuals prevention of new episodes in sudden cardiac arrest (SCA) survivals Relatives cascade screening of family members Further generations pre-implantation diagnostics

12 Sudden Cardiac Death PREVENTION Relatives Cardiac & genetic screening Affected individuals Asymptomatic carriers Genotypenegative Cardiological Follow-up Cardiological follow-up Excluded from Cardiological care

13 Comprenhensive Cardiac Panel Motol Cardiomyopathies Channelopathies Aortopathies Phenotype-like diseases Hypertrophic Cardiomyopathy (HCM) Dilated Cardiomyopathy (DCM) Long QT syndrome (LQT) Short QT syndrome (SQT) Thoracic aortic aneurysm and dissection (TAAD) Noonan syndrome Danon disease Arrhytmogenic right ventricular cardiomyopathy (ARVC) Restrictive cardiomyopathy (RCM) Left Ventricular noncompaction (LVNC) We have selected 229 genes related to heart and/or inherited cardiac disorders Cathecholamin ergic polymorphic ventricular tachycardia (CPVT ) Brugada syndrome (BrS) Marfan syndrome Loeys-Dietz syndrome Congenital contracturalara chnodactyly Arterial tortuosity syndrome Ehlers-Danlos syndrome Fabry disease Forbes disease Barth syndrome Pompe disease Friedrich s ataxia

14 Library preparation kit

15 WGS Best choice Big amount of data $$$$$ WES or clinical exomes Bigger amount of genes than a disease panel Higher data in WES than panels Might miss coverage of regions of interest $$$ Panels Better coverage of favorite genes Less data $

16 Pre-made panel No need to think which genes to include Validate / verify once (diagnostics) Custom panel Include your favorite genes Update and modify selected genes according to current literature and knowledge

17 When choosing a library prep kit take into consideration Genes included Transcript content (LRG ref seq, longest transcript, all transcripts) Target coverage Uniformity of coverage PCR duplicates (enrichment) Sequence quality Previous panel Current panel

18 Smaller or larger panel?

19 Why choose a broader custom panel? We may fill up runs faster increases TAT The increase in costs is low compared to the increase in data Research-mode and start up in a diagnostics setting include genes with clinical utility

20 Why choose a broader custom panel? Heterogeneity One gene Multiple genes Multiple diseases One disease ARVC MYOZ2 MYL2 MYLK2 TMPO GATAD1 TCAP CALR3 ABCC9 MYH6 MYL3 SGCD NEXN CAV3 RBM20 FHL2 EYA4 TNNC1 SDHA VCL LAMA4 PLN MYO6 FKTN LDB3 TPM1 JHP2 LMNA ACTN2 PSEN2 CSRP3 DMD SLC25A3 MURC TTN BAG3 PSEN1 MYPN SCO2 MYBPC3 CRYAB PRDM16 TRIM63 TNNI3 ACTC1 DSG2 MYH7 TNNT2 NEBL DSP DES JUP MIB1 DTNA PKP2 TAZ CTNNA3 RYR2 DSC2 RCM TGFB3 DCM HCM LVNC

21 Why choose a broader custom panel? We also may establish a proper diagnosis for rare genetic diseases which have similar phenotypic features. Patients referred as HCM patients to our Department (n = 352) Rare disease patients with HCM-like features (n = 10) HCM: Hypertrophic Cardiomyopathy

22 Variant Classification

23 ACMG guidelines /Genetic_Variant_Interpretation_Tool1. html/

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25 For HCM, the highest expected population frequency of the most common variant is filter-out variants with MAF > Ref. Whiffin et al., Genet Med Oct;19(10): doi: /gim

26 Hypertrophic Cardiomyopathy Re-assesment of variants Re-evaluation of variants in a selected group of patients - HCM with alcohol septal ablation treatment increase of data in population databases ACMG guidelines incorporation

27 Know your genes - FLAGS Genes with a high rate of rare (<1%) likely functional variants (missense, nonsense, splice site) data from the NHLBI GO Exome Sequencing Project (ESP)

28 Know your genes protein level Case 1. - Sudden death of a 14 yo boy from aortic dissection - Suspected Marfan syndrome - COL3A1 NM_ :c.2654G>A p.(gly885asp) triple helical tripeptide Gly-X-Y repeat region

29 Know your genes disease level Get information about the mechanism of the disease Which type of variants (null or missense) are disease causing? For example, for HCM MYBPC3 null variants (Myosin Binding Protein C, Cardiac) MYH7 missense variants (Myosin Heavy Chain 7)

30 Know your genes clinician level Get as much information as possible about the clinical phenotype of patients Case 2. - Female HCM patient severely affected for more than 10 years. - NGS cardio panel no significant variants - New information patient started to show signs of intellectual disability Danon disease? - Deep look at the LAMP2 gene - no significant variants - What about CNVs? Chr Pos Fold chance Pvalue Type Genes RoI name X E-04 Deletion (heterozygous) LAMP2 LAMP2_ex8 X Deletion (heterozygous) LAMP2 LAMP2_ex7 X Deletion (heterozygous) LAMP2 LAMP2_ex6 X E-05 Deletion (heterozygous) LAMP2 LAMP2_ex5 X E-04 Deletion (heterozygous) LAMP2 LAMP2_ex4 LAMP2 deletion of exons 4-8 GenesearchNGS, Phenosystems.

31 Variant 1 - MYBPC3 c.821+1g>a MYBPC3 is one of the most common mutated genes in HCM The c position disrupts a donor splice site Truncating mutations in MYBPC3 are known to cause HCM MAF-NFE: ; All = It segregates with 8 affected family members Reminder: do not forget splice variants in position +5 and synonymous variants in splice regions

32 Variant 2 - MYH7 c.4588c>t p.(arg1530*) MYH7 is one of the most common mutated genes in HCM MAF-NFE: ; All = Null variants in MYH7 are not know to be a cause of disease - two studies show MYH7 truncating mutations do not segregate with disease Healthy mother also carries the variant penetrance? Unknown significance class 3

33 Variant 3 - MYBPC3 c.146_148deltca p.(ile49del)

34 Variant 3 - MYBPC3 c.146_148deltca, p.(ile49del) MYBPC3 is one of the most common mutated genes in HCM MAF-NFE = Not in ClinVar HGMD disease causing mutation Found in two patients from a 696 Norwegian HCM patients cohort (Berge and Leren, Clin Genet Oct;86(4): ) Unknown significance class 3

35 Take home message

36 Take home message With time, whole-genome sequencing will be more affordable. For now, gene panels are a better choice for cardiac conditions Genetic testing by targeted-ngs panels allow us to properly identify cardiac conditions or phenotype-like disorders which is highly important for patient management Type of disease and financial capabilities are crucial when deciding if to use amplicon-based panels, enrichment panels, clinical exomes, WES or WGS

37 Take home message In diagnostics, it is important to adopt a standardized system for variant classification to assure quality results and decrease turn around time ACMG classification is a good candidate for variant classification Expanded genetic testing (broader panels) requires high strictness in variant interpretation. Therefore, a multidisciplinary approach is crucial for a proper molecular diagnosis To know your genes / proteins (or have expert s network) is highly important to perform correct classification this information affects patient s life

38 Acknowledgements Department of Biology and Medical Genetics, University Hospital Motol Prof. Milan Macek, MD, PhD Pavel Votypka Jan Geryk, PhD Tereza Rasplickova Michela Nemcikova, MD Alena Puchmajerova, MD Miroslava Balascakova, MD, PhD Veronika Zoubkova, MD Children s Heart Center, University Hospital Motol Peter Kubus, MD, PhD Veronika Stara, MD Institute of Inherited Metabolic diseases Lenka Piherova Cardiological Clinic University Hospital Motol Pavol Tomasov, MD, PhD Prof. Josef Veselka, MD, CSc Cardiological Clinic IKEM Alice Krebsova, MD, PhD

39 Questions?

40 NGS in Diagnostics: a practical example in hereditary cardiomyopathies Patricia Norambuena, PhD November 8th, 2017