ngs in diagnostics: a practical example in hereditary cardiomyopathies · 2017-11-14 · ngs in...
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NGS in Diagnostics: a practical example in hereditary cardiomyopathies
Patricia NorambuenaUniversity Hospital Motol, Prague.
2nd Faculty of Medicine, Charles University
VEP Course - November 6th - 8th, 2017
Sometimes the heart goes wrong…
From Loeys, 2016
Heart rhythm problems (arrhythmias / channelopathies)
Heart structural problems(cardiomyopathies)
Enla
rged
Rig
id
Thic
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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
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
Hypertrophic Cardiomyopathy
Private mutations
Population frequency: 1/500
Presents an age-related penetrance
Highly heterogenic phenotypeFrom asymptomatic patients to
Sudden Cardiac Death (SCD)
Hypertrophic Cardiomyopathy
Hypertrophic cardiomyopathy is
associated with mutations in sarcomere genes
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.
Hypertrophic Cardiomyopathy
Inherited cardiac conditions may lead
to
Sudden Cardiac Death (SCD)
Sudden Cardiac Death – PREVENTION
• prevention of new episodes in sudden cardiac arrest (SCA) survivals
Affected individuals
• “cascade” screening of family members
Relatives
• pre-implantation diagnostics
Further generations
Sudden Cardiac Death – PREVENTION
Relatives
Cardiac & genetic
screening
Affected individuals
CardiologicalFollow-up
Asymptomatic carriers
Cardiologicalfollow-up
Genotype-negative
Excluded from Cardiological
care
Comprenhensive Cardiac Panel Motol
Cardiomyopathies
Hypertrophic Cardiomyopathy
(HCM)
Dilated Cardiomyopathy
(DCM)
Arrhytmogenicright ventricular cardiomyopathy
(ARVC)
Restrictive cardiomyopathy
(RCM)
Left Ventricular non-compaction (LVNC)
Channelopathies
Long QT syndrome
(LQT)
Short QT syndrome
(SQT)
Cathecholaminergic
polymorphic ventricular tachycardia
(CPVT )
Brugadasyndrome (BrS)
Aortopathies
Thoracic aortic aneurysm and
dissection (TAAD)
Marfansyndrome
Loeys-Dietz syndrome
Congenitalcontracturalara
chnodactyly
Arterialtortuositysyndrome
Ehlers-Danlossyndrome
Phenotype-like diseases
Barth syndrome
Danon disease
Fabry disease
Forbes’ disease
Noonan syndrome
Pompe disease
Friedrich’s ataxia• We have selected 229 genes related
to heart and/or inherited cardiac
disorders
Library preparation kit
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
• $
“ 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
Current panel Previous panel
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
Smaller or larger 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
Why choose a broader custom panel?
Why choose a broader custom panel?
CALR3
CAV3FHL2
JHP2
ACTC1
ACTN2CSRP3
LDB3
ABCC9
EYA4
GATAD1
PSEN2
CRYAB
BAG3
DES
FKTN
RYR2
TGFB3
CTNNA3
JUPPKP2
DSC2
DSPDSG2
ARVC
DCMHCM
LAMA4
LVNC
DTNA
TAZ
PRDM16MYBPC3
TNNT2 NEBL
MIB1
MYL3
MYLK2
MYO6
MYOZ2MYL2
SLC25A3
TRIM63
SCO2
TPM1
TNNI3
TNNC1
TTN
VCL
TCAP
PLN
NEXN
MYH7
MYPN
MYH6
RCM
SGCD
SDHA
TMPO
MURC
LMNADMD
PSEN1
RBM20
• Heterogeneity
One gene
------------
Multiple genes
Multiple diseases
------------
One disease
• We also may establish a proper diagnosis for rare genetic diseases which have similar phenotypic features.
Why choose a broader custom panel?
Patients referred as HCM patients to our Department (n = 352)
Rare disease patients with HCM-like features(n = 10)
HCM: Hypertrophic Cardiomyopathy
Variant Classification
ACMG guidelines
http://www.medschool.umaryland.edu/Genetic_Variant_Interpretation_Tool1.html/
Ref. Whiffin et al., Genet Med. 2017 Oct;19(10):1151-1158. doi: 10.1038/gim.2017.26.
For HCM, the highest expected population frequency of the most common variant is 0.00004- filter-out variants with MAF > 0.0001
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
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)
Know your genes – protein levelCase 1.- Sudden death of a 14 yo boy from aortic dissection - Suspected Marfan syndrome- COL3A1 NM_000090.3:c.2654G>A p.(Gly885Asp)
triple helical tripeptideGly-X-Y repeat region
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)
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 119575575 -0.420942109 9.94E-04 Deletion (heterozygous) LAMP2 LAMP2_ex8
X 119576444 -0.526804924 0.006891 Deletion (heterozygous) LAMP2 LAMP2_ex7
X 119580150 -0.599590184 0.001013 Deletion (heterozygous) LAMP2 LAMP2_ex6
X 119581686 -0.518898753 1.68E-05 Deletion (heterozygous) LAMP2 LAMP2_ex5
X 119582815 -0.496193374 1.29E-04 Deletion (heterozygous) LAMP2 LAMP2_ex4
GenesearchNGS, Phenosystems.
LAMP2 deletion of exons 4 - 8
Variant 1 - MYBPC3 c.821+1G>A
MYBPC3 is one of the most common mutated genes in HCM
The c.821+1 position disrupts a donor splice site
Truncating mutations in MYBPC3 are known to cause HCM
MAF-NFE: 0.000014; All = 0.000030
It segregates with 8 affected family members
Reminder: do not forget splice variants in position +5 and
synonymous variants in splice regions
Variant 2 - MYH7 c.4588C>T p.(Arg1530*)
MYH7 is one of the most common mutated genes in HCM
MAF-NFE:0.000018; All = 0.000030
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
Variant 3 - MYBPC3 c.146_148delTCA p.(Ile49del)
Variant 3 - MYBPC3 c.146_148delTCA, p.(Ile49del)
MYBPC3 is one of the most common mutated genes in HCM
MAF-NFE = 0.00002794
Not in ClinVar
HGMD – disease causing mutation
Found in two patients from a 696 Norwegian HCM – patients cohort (Berge and Leren, Clin Genet. 2014 Oct;86(4):355-60.)
Unknown significance – class 3
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
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
Take home message
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
Cardiological ClinicUniversity Hospital Motol• Pavol Tomasov, MD, PhD• Prof. Josef Veselka, MD, CSc
Cardiological ClinicIKEM
• Alice Krebsova, MD, PhD
Children´s Heart Center, University Hospital Motol• Peter Kubus, MD, PhD• Veronika Stara, MD
Institute of Inherited Metabolic diseases• Lenka Piherova
Acknowledgements
Questions?
NGS in Diagnostics: a practical example in hereditary
cardiomyopathies
Patricia Norambuena, PhDNovember 8th, 2017