Genetic testing in Inherited Cardiac Conditions (ICCs)

Question 1

Inherited cardiac conditions involve what?

Answer 1

Diseases of the heart and circulation

Question 2

What is the combined prevalence of Inherited cardiac conditions?

Answer 2

1%

Question 3

What do inherited cardiac conditions include?

Answer 3

Cardiomyopathies
Cardiac arrhythmias
Aortopathies
Hyperlipidaemias
Congenital cardiac conditions

Question 4

Inherited cardiac conditions are a very common cause of sudden death at all ages

True or false

Answer 4

True

Question 5

Inherited cardiac conditions are most commonly autosomal dominant, but variable expressivity and penetrance

True or false

Answer 5

True

Question 6

Cardiac Conditions are at the interface between…

Answer 6

Rare and complex disorders

Question 7

Inherited disorders caused by different genetic constellations

What are some examples?

Answer 7

1. Single gene, single major mutation
   - α-1-antitrypsin deficiency (AAT): SERPINA1, Glu342Lys mutation (Z-allele)
   - Haemachromatosis: HFE, C282Y (83% of patients)
2. Single gene, many mutations
   - Cystic fibrosis (CF): >2000 mutations, F508del most common
   - Marfan syndrome (MFS): >1000
3. Many genes, many mutations
   - Cardiomyopathy: 11 core genes (+ many more), 30—50% patients

Question 8

Most inherited disorders are rare, even the well-known ones.

What are some examples?

Answer 8

Cystic Fibrosis
Duchenne Muscular dystrophy
Fragile X-linked intellectual disability
Huntington’s Disease
Marfan’s Syndrome

Question 9

Most complex diseases have severe, inherited, monogenic forms

What are some examples?

Answer 9

Dilated Cardiomyopathy

Hypertrophic Cardiomyopathy

Question 10

The development of sequencing technology involve what?

Answer 10

ABI 3500: For Single gene sequencing (Sanger)

MiSeq: Targeted analysis using multiple (small) gene panels (Targeted NGS)

NextSeq:

Targeted analysis using multiple (large) gene panels

Whole exome sequencing (WES)

HiSeq: Whole Genome Sequencing (WGS)

Question 11

What do genetic anomalies of Inherited Cardiac Conditions involve?

Answer 11

Common, serious, actionable

Often late-onset, not many large families

Allelic heterogeneity

High burden of rare variants

Many unclassified variants (VUS)

Bi-allelic mutations, despite autosomal dominant inheritance

Incomplete penetrance

Modifiers

Double (di-genic) mutations

Cascade genetic testing (testing family members) is now Class 1 indication (recommended)

Incidental findings (a problem with large panels)

Question 12

Define Expressivity

Answer 12

Quantifies variation in a non-binaryphenotypeacross individuals carrying a particulargenotype.

Variable expressivity occurs when a phenotype is expressed to a different degree among individuals with the same genotype.

Question 13

Define penetrance

Answer 13

The proportion of people with a given genotype who will exhibit symptoms of a condition

Question 14

What is Dilated cardiomyopathy (DCM)?

Answer 14

Dilation of the (left) ventricle, affecting the heart’s pumping ability
(~30% has a genetic cause)

Question 15

What is hypertrophic cardiomyopathy (HCM) ?

Answer 15

HCM (in the absence of hypertension or aortic stenosis):

Heart muscle becomes thickened (hypertrophy) and stiff, and cells become disarrayed

Question 16

There is a dramatic increase in number of rare disease genes identified and gene-disease associations.

However not all…

They are not always accompanied by what?

Answer 16

Not all new associations have robust evidence - strong segregation or clear excess burden of variants in disease vs control cohorts

Not always accompanied by functional studies

Question 17

What are known core genes associated with diseases?

Answer 17

Long QT syndrome: KCNQ1 (40-55%) , KCNH2 (35-45%), SCN5A (2-8%)

Brugada Syndrome: SCN5A (20%)

CPVT: RYR2 (50%)

HCM: MYBPC3 (20- 30%), MYH7 (15-20%), TNNT2 (5%). TNNI3 (5%)

DCM: TTN (15-20%), MYH7 (5%) , LMNA (5%) , TNNT2 (2 3%).

Question 18

What do allele frequencies: Gene burden analysis using (very) large cohorts involve?

Answer 18

Compare allele frequencies of rare variants (MAF<0.0001) in healthy control cohorts with patient cohorts

If excess in patients, indication for association with disease

Re-evaluation of genes previously implicated in Mendelian disease

Allows accurate evaluation of pathogenicity of different classes of variants

Particularly useful for newly-associated genes and new findings

Question 19

What is Genome Aggregation Database (gnomAD)?

Answer 19

Collection of whole-exome and genome sequencing data from ~125,000 exomes and ~15,700 genomes; mostly healthy individuals

Represents diverse human populations:
European (Finnish and non-Finnish separated), African, Latino, South Asian, East Asian, “other”

Data released (exomes only ‘ExAC’) late 2014 (Lek M et al Nature 2016)

Phenotype data for individuals not initially included, but available on request

Individuals affected by severe paediatric disease excluded

Comparison of carrier allele frequencies for well-known inherited disorders, e.g. cystic fibrosis demonstrated that database not over-enriched for pathogenic variants (Song W et al. Genet Med 2016)

Utility as a control cohort for genetic analysis

Question 20

What Population “Control” Variant Databases include cardiac disease samples

Answer 20

ExAC

Esp

Question 21

What Population “Control” Variant Databases include the general population?

Answer 21

1000 Genomes

Question 22

What Population “Control” Variant Databases include Phenotypically –defined healthy?

Answer 22

HVOLS

Question 23

Why should literature be questioned?

Answer 23

Presence of a variant in a patient with disease DOES NOT always imply causation (ie: don’t blindly believe literature / HGMD)

Earlier publications did not have access to population databases now available:
E.g. MYH6: p.R1502Q – detected in 1/312 DCM patients, absent in 246 controls

Question 24

What can validate genes role, e.g. cardiomyopathies?

Answer 24

Unbiased analysis using large disease and control (ExAC) cohorts

Question 25

Genetic diagnosis of Inherited Cardiac Conditions (ICCs) | involves what genes?

Answer 25

All ICC = 169 genes Cardio-myopathy genes (92) Congenital ICCs (19) Arrhythmia genes (38) Aortopathy genes (20)

Question 26

What does the diagnostic procedure/ LIMS (Laboratory Information Management System) involve?

Answer 26

``` Blood/sample received in lab with consent DNA extraction Sequencing library prep Next-Gen Sequencing 169 genes Bioinformatic analysis Sanger confirmation Report ```

Question 27

Describe the bioinformatics pipeline

Answer 27

1. Sequencing, this results in a Fastq file 2. FastQC, QC 3. Reference human genome, mapping, results in SAM/BAM file 4. Preprocessing, picard and Samtools 5. Variant calling, GATK (Unified Genotyper/ Haplotype Caller. Results in VCF file containing SNVs and Indels 6. Variant filtering and annotation 7. Database upload

Question 28

What are two types of genetic tests?

Answer 28

Mutation testing first pursued whereby an NHS gene panel is used and next depending on findings from this, other investigations can be pursued in form of cascade testing. Mutation screening involves an NHS gene panel, if a pathogenic variant is found targeted analysis of a single familial variant (Sanger seq/ MLPA/ddPCR), diagnosis and predictive testing may then be followed. If VUS segregation analysis pursued.

Question 29

Why is variant assessment and interpretation important?

Answer 29

Limiting factor shifted from acquisition of sequence data to classification, interpretation and reporting of DNA variants Accurate interpretation has not kept pace with increasing amount of sequence data Most novel and recurring sequence variants: little/no conclusive information regarding disease causation Recognition: phenotypic spectrum of many disorders larger than appreciated Targeted Panel design is important - Single genes can be causative for many disorders

Question 30

What is a VUS? How often are they found? What is the impact on disease?

Answer 30

Variants that haven't been seen before (or only very rarely) In our service: ~25% of cases are reported with one Impact on disease isn't clear

Question 31

If a VUS is found what should next occur?

Answer 31

Re-classification possible Further segregation analysis in families More patients with the variant described Functional analysis at the RNA or protein level

Question 32

Should a VUS be used in clinical decision making?

Answer 32

No

Question 33

What guidelines aid variant interpretation?

Answer 33

ACMG

Question 34

Why is congregation difficult to pursue? What should be considered?

Answer 34

Requires availability of appropriate family samples - No material available from patients after sudden cardiac death - Large families usually not available (late-onset conditions) - Type of cardiac death in older generations often unclear Consider possibility of phenocopies and partial penetrance

Question 35

What is cosegregation?

Answer 35

Gold Standard in variant interpretation – full segregation in large family provides near definitive evidence of variant’s role in causing disease.

Question 36

What happens after genetic test results?

Answer 36

If a disease-causing (pathogenic) mutation is identified: - Clinician needs to consider if this provides the “whole explanation” for patient and or family. - It may confirm the clinical diagnosis - It may affect treatment or patient management - Allows accurate estimate of recurrence risks - Allows carrier and predictive testing of at risk family members

Question 37

What are the implications of genetic testing in Inherited Cardiac Conditions (ICCs)?

Answer 37

Single gene analysis results in low mutation yield for most cardiac conditions Targeted Panel diagnostics increases the mutation yield WES and WGS may increase information e.g. about modifiers Molecular genetic diagnosis directs (increasingly in the future) clinical cardiological surveillance Known pathomechanism enables targeted therapy (eg: Selumetinib, an inhibitor of the ERK1/2 signalling pathway investigated for cancer trials, preserves cardiac function in mouse models with DCM caused by LMNA variants