Genetic testing in Inherited Cardiac Conditions (ICCs) Flashcards
Inherited cardiac conditions involve what?
Diseases of the heart and circulation
What is the combined prevalence of Inherited cardiac conditions?
1%
What do inherited cardiac conditions include?
Cardiomyopathies Cardiac arrhythmias Aortopathies Hyperlipidaemias Congenital cardiac conditions
Inherited cardiac conditions are a very common cause of sudden death at all ages
True or false
True
Inherited cardiac conditions are most commonly autosomal dominant, but variable expressivity and penetrance
True or false
True
Cardiac Conditions are at the interface between…
Rare and complex disorders
Inherited disorders caused by different genetic constellations
What are some examples?
- Single gene, single major mutation
- α-1-antitrypsin deficiency (AAT): SERPINA1, Glu342Lys mutation (Z-allele)
- Haemachromatosis: HFE, C282Y (83% of patients) - Single gene, many mutations
- Cystic fibrosis (CF): >2000 mutations, F508del most common
- Marfan syndrome (MFS): >1000 - Many genes, many mutations
- Cardiomyopathy: 11 core genes (+ many more), 30—50% patients
Most inherited disorders are rare, even the well-known ones.
What are some examples?
Cystic Fibrosis Duchenne Muscular dystrophy Fragile X-linked intellectual disability Huntington’s Disease Marfan’s Syndrome
Most complex diseases have severe, inherited, monogenic forms
What are some examples?
Dilated Cardiomyopathy
Hypertrophic Cardiomyopathy
The development of sequencing technology involve what?
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)
What do genetic anomalies of Inherited Cardiac Conditions involve?
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)
Define Expressivity
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.
Define penetrance
The proportion of people with a given genotype who will exhibit symptoms of a condition
What is Dilated cardiomyopathy (DCM)?
Dilation of the (left) ventricle, affecting the heart’s pumping ability
(~30% has a genetic cause)
What is hypertrophic cardiomyopathy (HCM) ?
HCM (in the absence of hypertension or aortic stenosis):
Heart muscle becomes thickened (hypertrophy) and stiff, and cells become disarrayed
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?
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
What are known core genes associated with diseases?
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%).
What do allele frequencies: Gene burden analysis using (very) large cohorts involve?
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
What is Genome Aggregation Database (gnomAD)?
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
What Population “Control” Variant Databases include cardiac disease samples
ExAC
Esp
What Population “Control” Variant Databases include the general population?
1000 Genomes
What Population “Control” Variant Databases include Phenotypically –defined healthy?
HVOLS
Why should literature be questioned?
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
What can validate genes role, e.g. cardiomyopathies?
Unbiased analysis using large disease and control (ExAC) cohorts
Genetic diagnosis of Inherited Cardiac Conditions (ICCs)
involves what genes?
All ICC = 169 genes
Cardio-myopathy genes (92)
Congenital ICCs (19)
Arrhythmia genes (38)
Aortopathy genes (20)
What does the diagnostic procedure/ LIMS (Laboratory Information Management System)
involve?
Blood/sample received in lab with consent DNA extraction Sequencing library prep Next-Gen Sequencing 169 genes Bioinformatic analysis Sanger confirmation Report
Describe the bioinformatics pipeline
- Sequencing, this results in a Fastq file
- FastQC, QC
- Reference human genome, mapping, results in SAM/BAM file
- Preprocessing, picard and Samtools
- Variant calling, GATK (Unified Genotyper/ Haplotype Caller. Results in VCF file containing SNVs and Indels
- Variant filtering and annotation
- Database upload
What are two types of genetic tests?
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.
Why is variant assessment and interpretation important?
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
What is a VUS?
How often are they found?
What is the impact on disease?
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
If a VUS is found what should next occur?
Re-classification possible
Further segregation analysis in families
More patients with the variant described
Functional analysis at the RNA or protein level
Should a VUS be used in clinical decision making?
No
What guidelines aid variant interpretation?
ACMG
Why is congregation difficult to pursue?
What should be considered?
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
What is cosegregation?
Gold Standard in variant interpretation – full segregation in large family provides near definitive evidence of variant’s role in causing disease.
What happens after genetic test results?
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
What are the implications of genetic testing in Inherited Cardiac Conditions (ICCs)?
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
