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

k

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?

patricia.norambuena@fnmotol.cz

NGS in Diagnostics: a practical example in hereditary

cardiomyopathies

Patricia Norambuena, PhDNovember 8th, 2017