New Technologies in

Genetic Testing II: NGS

BY: BIANCA CARZIS
MSC (MED) GENETIC COUNSELLING
Objectives
By the end of this lecture you should be able to:
1. Distinguish between disease-targeted gene panels, whole exome sequencing (WES) and
whole genome sequencing (WGS)
2. Describe some of the benefits and limitations of NGS applications in the clinical context.
3. Identify the challenges faced in a clinical diagnostic setting when offering NGS testing.
4. Justify the uses of and choices made in deciding which NGS application to employ in specific
clinical scenarios.
5. Discuss the role of the genetic counsellor/clinician in advising and interpreting the results
from NGS in clinical applications.
Clinical NGS applications
Next generation sequencing
Sanger Sequencing à Next Generation Sequencing

• Increased
• Directed/targeted
resolution
• Family
• Less
specific/common
specific/targeted
mutations
• Relatively cheaper
• Accurate
(but still
• Costly
expensive)
• Time consuming
• Rapidly advancing
NGS Applications
Single gene analysis (10-50kb)

Sequencing panels (1Mb)

Exome sequencing (50Mb)

Genome sequencing (3000Mb)

Targeted Gene Panel Testing
◦ Analyse multiple ‘related’ genes simultaneously
◦ May be 2-100+ genes
◦ High yield if carefully selected
◦ Less ambiguous results
◦ Less unwanted results
◦ Useful if:
◦ Phenotype relatively distinct (e.g. muscular dystrophy, breast cancer)
◦ Multiple genes known to cause similar phenotype
◦ Locus heterogeneity
◦ Cannot distinguish causative genes based on phenotype
 Whole Exome Sequencing (WES)
Sequence entire coding sequence of human genome (exons)
◦ Analyse 1-2% of the genome BUT 85% of pathogenic mutations in exons
◦ Less targeted approach
◦ Useful to identify new genes involved in pathogenesis
◦ Increased chance of uncertain findings
Consider if:
◦ Poorly defined phenotype
◦ Suspected new syndrome
Clinical exome concept:
Interpretation challenging ◦ Subset of exome
◦ Often generate new data ◦ Genes with known clinical
◦ Difficult to prove causation significance
◦ Negative panel?
Whole Genome sequencing (WGS)
Sequence all DNA of an individual
(3000Mb)
Massive data set
◦ Very large number of variant
May detect:
◦ Deep intronic mutations
◦ Breakpoints
◦ Structural rearrangements
Increased chance of uncertain
findings
Mainly used in research setting
Choosing the Right Genetic Test Traditional methods are slow
and expensive but very
Has testing been performed accurate.
previously? Which genes are included in
Limitations
Is there a known mutation in the of genetic
the panel?
family? testing What is their ”actionability”?
approaches
What is the coverage of the
panel?
Is Sanger sequencing used to
confirm mutations?
Genetic
Family
history Test Ethnicity
Decisions

Interpretation of genetic testing is Is the patient from a high risk ancestry group?
complicated. Is there testing available for the common
Is there a more appropriate family mutations found in this ethnic group?
Who to test
member to test? What is the pick up rate of this test i.e. how
Is there more than one person who many people who have the disease will test
should be tested? positive using this test?
Challenges and
limitations
 NGS Challenges
A typical WES sample will have 10 000 – 20 000
variants
A typical WGS sample will have ± 3 million variants

Analysis is complex
• Requires expertise
• Interpretation Influenced by experience &
Knowledge of individual genes
• Discrepancies between labs

Store? Visualise? Manipulate? Analyse? Integrate?

HOW DO WE MAKE IT RELEVANT AND APPLICABLE TO THE PATIENT?

Variants of Uncertain Significance
An alteration in the normal sequence of a gene, the
significance of which is unclear
•Knowledge limitations
•Classification of variants complex
◦ Bioinformatic analysis
◦ Database knowledge
◦ Lack of African data
•Additional studies may prove it to be benign or disease-
causing
◦ Family segregation studies
◦ Functional studies
•Translation for patient?
Incidental findings
Incidental or secondary findings
◦ Unrelated to the indication for testing
◦ May be of medical value for patient care
◦ Not part of reason for test
◦ Counselling issues
◦ When to inform
◦ Reportable/actionable variant
◦ 1% of tests have a reportable incidental variant
◦ ACMG list of 59 genes
◦ Pathogenic and likely pathogenic variants must be reported to patients e.g. inherited
cancer, cardiomyopathy, familial hypercholesterolaemia, Marfan syndrome
 Other Challenges
Missing heritability
◦ Missed mutations – challenges of a negative test
◦ Larger rearrangements
◦ Technical issues
◦ Epigenetic changes
◦ Other ?
Genetic complexity (heterogeneity)
◦ Gene mutations in different genes cause same disease
◦ Mutations in same gene cause different diseases
◦ Pathways – modifiers
The role of the genetic
counsellor/clinician
Adapting Genetic Counselling for the Genomic
Era

“…the ability to interpret data and translate the data

into clinically meaningful information for patients,
the ability to explain genetic technologies to patients,
the ability to communicate uncertainty to patients
(e.g. variants of uncertain significance results),
and the ability to know how much and which
information to share with patients”

Profato et al (2014) Assessing the integration of

genomic medicine in genetic counselling training
programs. J Genet Counsel, 23:670-688
 Adapting Genetic Counselling for the Genomic Era
The scale of information provided by genomic sequencing is vast
◦ How do we determine which results to return and when to return them?
A more critical evaluation of genetic variants and their association with disease risk is required:
◦ Primary literature
◦ Online and other electronic tools
Strong focus on informed consent particularly with respect to:
◦ Uncertainty
◦ Ownership of genetic data
◦ Duty to re-contact and/or re-annotate
Case Studies
Case 1: Use Of NGS Muscle Panel
Clinical Evaluation:
Male with degenerative muscle disease
◦ Initially thought to be Becker Muscular Dystrophy
◦ Tested negative with single gene test (detects 90%+ of mutations)
◦ Seen when muscular dystrophy had progressed too far for muscle biopsy to be useful
◦ Clinical diagnosis uncertain
◦ Sisters concerned about risk of having similar affected children
o X-linked recessive - ? risk
o Autosomal recessive – ? risk
o New autosomal dominant – ? low risk

Genetic testing:
No clear indication which muscular disorder gene to test
◦ Opted for broad muscle disease panel (>150 genes)
◦ All genes associated with different muscular dystrophies
Case 1:Use Of NGS Muscle Panel
Result:
Two different mutations identified in GNE gene
◦ DIAGNOSIS: Inclusion body myopathy
◦ Features consistent
◦ Autosomal recessive inheritance

Outcomes:
What risk do you give the sisters? Why?
 Case 2: Use Of Exome Sequencing
Clinical evaluation:
•Three affected individuals in doubly consanguineous family
•Non-progressive myoclonic epilepsy, progressive cerebellar ataxia, normal cognition and sensation, childhood
onset, in wheelchairs by teens
•Clinical diagnosis uncertain
◦ Unable to find other cases in literature with similar phenotype

Genetic testing:
Exome sequencing
◦ What inheritance pattern are you expecting?
.
 Case 2: Use Of Exome Sequencing
Result:
Identified a homozygous mutation c.430G>T, p.Gly144Trp in GOSR2 gene
Outcome:
Rare condition – first described in 2011
◦ 10 families known worldwide
◦ All with identical homozygous mutation
◦ All from North Sea area
◦ Phenotype still not fully described
South African family
◦ Had epilepsy treated with deep brain stimulation
◦ Significant positive effect on ataxia (shown by switching on and off DBS under controlled conditions)
◦ Offers potential new method of managing GOSR2 patients
◦ Contribution to scientific literature
 Case 3: Unexpected information
Clinical evaluation:
Family with two children with unusual cancers
•Child was diagnosed with a neuroblastoma at the age of 10 months (died at 20 months)
•Second child, was diagnosed with a brain tumour
•Parents healthy and no family history of cancer
Genetic testing:
Child tested on cancer panel – 83 genes
Results:
Two pathogenic variants,
Inconsistent with family hx
Case 3: Unexpected information
Outcomes:
Father has p53 mutation
◦ Recently diagnosed with prostate cancer at age 49
◦ Parents healthy - >70 years
Mother has BRCA2 mutation
◦ No family history
Unaffected sibling
◦ 18 years old
◦ Carries BOTH mutations
◦ Surveillance
◦ ? Interaction of two mutations