Ngs presentation

Whole Exome and Genome sequencing
in Neurological disorders

•Introduction to Genetics
•Definitions
•Technique of NGS
•Clinical Application
•Limitations
•Conclusion
Over View
Whole Exome and Genome sequencing in Neurological disorders

•Human genome is a complete set of nucleic acid sequence encoded
as DNA with in 23 pairs of chromosomes and Mitochondrial DNA
•The total length of the human genome is over 3 billion base pairs
Gene
A Gene is a locus (or region) of DNA which is made up of nucleotides
and is the molecular unit of heredity
The Human Genome Project has estimated that humans have
between 20,000 and 25,000 genes.
Introduction

Each Gene consists of Regulatory component and Reading Frame
•Regulator region includes -Enhancer/silencer and
-Promoter/Terminator
Reading Frame includes – Exons and Introns
• Exon is a segment of gene that codes for protein
• Intron is the non protein coding segment that exists b/w Exon
segments with in reading Frame
• Exome includes all the protein coding segments in entire genome
• In humans Exome constitutes only about <2% of the Genome
Introduction

Introduction
Structure of a GENE

DNA sequencing has started with 2 dimensional Chromatography in
1970s that could read only 4-6 base pair length
Generation 1-Manual sequencing
They used in vivo cloning by using plasmids, targeted DNA sequences
•In 1977 Sanger Sequencing (Radio labelled nucleotide chain
termination) was started
• Maxam Gilbert method( chemical chain termination)
These techniques could sequence DNA of only 700 bases length in a
cycle
DNA sequencing Techniques

Generation 2-Automated sequencing
In 1987 Automation of Sanger Sequencing was developed, sequencing
became faster but read length remained same
Generation 3 –Next Generation Sequencing(NGS)
It involves amplification of DNA in vitro using PCR technique to create
clusters of long segmented DNA that could be sequenced
In 2005 First genome analyser was developed using this 1 Giga base pair
could be sequenced per run compared to 84 kilo base pairs per run
using older methods

Methods for genetic diagnostic testing
Single-gene sequencing
Testing for a mutation in a specific gene that is known to be
associated with a particular disease
• Sanger sequencing technique is used
•In clinical practice, the clinician must determine the most
probable disorder and order testing of the specific gene(or genes)
that causes the illness.
•It does not detect large insertions/deletions, copy number
variations, or nucleotide repeat expansions and also not
applicable for testing of all genes
•The cost of Sanger sequencing - $1,000 per gene have been
suggested

Multigene sequencing (Sanger sequencing, panel testing)
•Genetic panels of variable size for testing multiple genes
simultaneously are useful in clinically heterogeneous neurologic
disorders (e.g., intellectual disability, epilepsy, ataxia, leukodystrophy,
polyneuropathy),
•These panels commonly utilize Sanger sequencing methods
•Commercial price can be expensive (e.g., $30,000 or more)

Chromosomal microarray analysis
•It is a technique for detecting clinically significant structural changes,
particularly deletions or duplications in the genome, copy number
variation
•As WES/WGS cannot detect sequence variation, CMA is used
complementary in NGS to detect structural variation that
Exome/Genome sequencing cannot

•First whole genome sequencing
•It started in 1990 and completed in 2003
• Costed 3.8 billion$
•It used 1st and 2nd generation techniques
HUMAN GENOME PROJECT

DEFINITION
Next-Generation Sequencing(NGS)
Any process that involves rapid sequencing of large amount of DNA
segments up to and including entire genomes
Concept of rapid sequencing of large segment of DNA can be used to
sequence
•Entire Genome
•Entire exome
•Any DNA segment of interest
Accordingly there are several applications of NGS
Sequencing of entire Genome /Exome is primarily used in clinics

Whole-Genome Sequencing
Determination of the sequence of most of the DNA content
comprising the entire genome of an individual.
•In a present-day “whole-genome sequence (WGS) certain
components of the genome may not be sequenced.
Whole-Exome Sequencing
Determination of the sequence of DNA that encodes protein-
Exons and may include some DNA regions that encode RNA
molecules that are not involved in protein synthesis.
•Though Exome represents less than 2% of the genome it
contains most of the disease causing variants which makes it a
cost effective alternative to whole genome sequencing
DEFINITION

Whole Exome Sequencing Whole Genome Sequencing
Advantages
•Sequencing of clinically
relevant genes with complex
structure is better
•Less cost
•Over all sequencing of coding
region is 98% compared to 95% by
WES
•Variants in non coding regions
can be identified
Disadvantages
•Does not cover entire genome
•Sequencing bias are more
•Large number of variants are
generated
•Comparatively more no of genes
are missed and it depends on the
platforms used for sequencing

NGS experiments consist of 4 phases
•Sample collection
•Template generation
• Amplification
•Sequencing
•Alignment and data analysis
Technique of NGS

Sample collection
Technique of NGS

By fragmenting the
sample dsDNA Templates
are generated, these
Templates are ligated to
Adaptors which act as
primers for DNA synthesis
The fragmented DNA segments attached to Adaptors form NGS library
Technique of NGS
Template generation and Amplification

NGS library is loaded
on to a platform called
flow cell , here the
fragments are fixed
according to the size
These bound fragments are cloned by using fluorescently labelled
neuclotides through bridge amplification to form clusters
Technique of NGS
Template generation and Amplification

Sequencing
The emission from each
cluster is recorded
Depending on the
wavelength and
intensity of emission
nitrogen bases are
identified and are read
Technique of NGS

Technique of NGS
Using the bioinformatics soft
ware the identified bases are
aligned into a sequence
Difference b/w the reference Genome and newly generated sequence
can be identified
Alignment and data analysis

Benefits of accurate molecular diagnosis
•Limits the unnecessary and invasive investigations
•Ineffective and potentially harmful treatment can be
avoided
•Helps in determining methods for monitoring of the
disease
•Pre symptomatic family members can be identified
•Provide accurate recurrence risk
•Helps in counselling for prenatal and preconception
options
•Provide psychological benefits to patients and family
members by knowing the cause of the disease
Clinical application

Indications of NGS
•Disorders with genetic heterogeneity
•Genetic disorders with phenotypic variability
•Failure of Monogenic testing in a suspected Genetic disorder
•Prenatal diagnosis of Suspected genetic Disorder when routine
work up has failed
•Disorders caused due to multiple genes involvement

Neurological presentations of genetic disorders
involve the full range of the neuroaxis and include
•Structural malformations of brain,
•Epilepsy syndromes,
•Neurometabolic disorders,
•Ataxias,
•Encephalopathies,
•Myopathies and Muscular dystrophies,
•Neuropathies,
•Movement disorders, and
•Dementias

A total of 191 cases with leucoencephalopathies were collected
from August 1, 2009 to July 31, 2013 in
The Myelin Disorders Bioregistry Project(MDBP) or
The Amsterdam Database of Unclassified Leukoencephalopathies .

•101 patients were diagnosed using MRI pattern recognition
followed by biochemical or other molecular approaches
• Of the 90 undiagnosed cases 71 under went WES
• Diagnostic pathogenic variants were identified in 35% (25 of 71),
Potentially pathogenic variants in 7% (5 of 71) of patients.
• A total yield of clinical diagnoses in 42% of individuals
Conclusion by the author:
Majority of disorders associated with abnormal white matter on
neuro imaging are not classic leukodystrophies. This suggests that
testing of only leukodystrophy associated genes on NGS panels
may have limited diagnostic efficacy (predicted to be only 13% in
this cohort

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous
group of disorders, To date, 27 different genetic forms of
LGMD(types and subtypes) have been described and are grouped
according to inheritance pattern
237 patients with LGMD were retrospectively selected through the
Institute for Neuroscience and Muscle Research Biospecimen Bank
(between 2006 and 2014),

Based on clinical indications, immunohistochemistry,
candidate genetic testing was performed

Of the 154 families with undiagnosed LGMDs, 62 satisfied the
inclusion criteria,60 consented for WES
With WES-
•Pathogenic mutations in known myopathy genes in 27 families
•Mutations in known LGMD-related genes in 12 families
• Variants in disease-related genes not typically associated with
LGMD in 15 families
•Over all efficacy of WES was about 60%(Inclusion of family
members increased the diagnostic efficacy )

In the remaining undiagnosed families,
•Disease-causing mutations may be located in non coding regions of
genes, such as regulatory or deep intronic regions not captured and
sequenced by use of WES.
•Mutations in coding regions of known myopathy-related genes may
have been missed by both methods of NGS because 5% to 10% of
coding exons are poorly captured by a range of NGS methods.
•The presence of repetitive regions/ copy number variants
•Deletions that remove entire exons.

Cerebellar ataxias are a diverse collection of neurologic disorders
Numerous genetic disorders have been associated with chronic
progressive ataxia
Series of 76 patients presenting to a tertiary referral centre for
evaluation of chronic progressive cerebellar ataxia were
included in the study and underwent WES

Adult onset in 72%, Sporadic-onset 74%
•Known Pathogenic mutations were detected in 16
patients(21%)
38% (6 of 16) were adult-onset
69% (11 of 16) presented sporadically
•Variants with potential pathogenicity were detected in 30
patients (40%) 77% (23 of 30) were adult-onset
73% (22 of 30) presented sporadically.
• In 30 of the 76 cases (40%) no pathogenic mutation was
identified
CONCLUSION: In the evaluation of patients with early/adult-
onset ataxia, with or without a family history, in the presence of
an otherwise non-diagnostic clinical workup, NGS may further
improve the diagnostic outcome

All patients with intractable epilepsy and GDD and cognitive
Dysfunction seen between January 2012 and June 2014 in a
single epilepsy genetics clinic at The Hospital for Sick Children
(Toronto, Canada) were included in this retrospective cohort
study

110 patients were included, 93 patients had no recognizable
syndromic clinical features, MRI,(MRS) patterns, metabolic
investigations, or aCGH abnormalities . They underwent Targeted
NGS for epileptic encephalopathy using various gene panels
•Targeted next-generation sequencing identified a genetic cause in
28% of the patients.
•The lower diagnostic yield likely depends on the small number of
the genes included in those panels

Targeted next-generation sequencing panels increased the genetic
diagnostic yield from 10% to 25% in patients with epileptic
encephalopathy.
Conclusion: Targeted NGS is recommended for the identification of
underlying genetic causes of epileptic encephalopathy in patients with
• Normal first line biochemical investigations,
•Normal aCGH, no seizure response to pyridoxine or pyridoxal-5-
phosphate, and
•No recognizable syndrome or Characteristic brain MRI changes to point
out to a specific genetic disorder.

Epilepsies have a highly heterogeneous background with a strong
genetic contribution, a variety of unspecific and overlapping
syndromic and non syndromic phenotypes often hampers a clear
clinical diagnosis and prevents straightforward genetic testing.

• 33 cases with different epilepsy phenotypes were randomly
selected from various hospitals and clinical practices in
Germany and Switzerland
•265 genes that were known to be involved in monogenic
disorders including epilepsy as a phenotypic feature according
to the Online Mendelian Inheritance in Man (OMIM) database
were grouped into genetic epilepsy panel and was applied to
each patient
•Disease-causing mutations were diagnosed in 16 of 33
patients (48%)

Conclusion:
•Disease-specific gene panel allows a significant increase of
coverage on target sequences
• This approach is only suited in diagnostics of monogenic
disorders.
•WES/WGS is beneficial in complex genetic disorders, as it
has the advantage of covering all coding genes

•Mitochondrial disorders have both genotypic and phenotypic
heterogeneity
•109 patients at Nijmegen Centre for Mitochondrial Disorders
(NCMD) between December 2011 and June 2013 were included
•All patients first under went sequencing using a Genetic panel
that included of 238 mitochondrial specific genes, later WES was
performed

•A pathogenic mutation explaining the phenotype was found in
16 patients using genetic panel, with WES 42 patients were found
to have pathogenic mutations
CONCLUSION: WES is beneficial particularly in those with
inconclusive findings on routine work up for mitochondrial
disorders
•Genetic panel can be used when a single or few specific
disorders are highly suspected after regular work up

•Intellectual disability is a genetically heterogeneous disorder,
more than 1000 different genes may cause intellectual disability
•50 Patients with severe ID (IQ <50) and No significant
Diagnostic clues on metabolic and genetic screening with
genomic microarrays and exome sequencing, underwent WGS
along with unaffected family members

pathogenic causative mutations was identified in 42%(21 of 50)
patients by WGS
Conclusion: Genome sequencing can be applied as a single
genetic test to reliably identify and characterize the
comprehensive spectrum of genetic variation, providing a
genetic diagnosis in the majority of patients with severe ID

Genetic heterogeneity in
hereditary peripheral
neuropathy

•It was a retrospective review of samples from 103 patients
investigated for hereditary peripheral neuropathy, received by
Telemark Hospital in the period 2012 – 14.
•Prior to NGS, 7 patients were diagnosed with duplication or
deletion of PMP22 via Multiplex Ligation Probe Amplification
(MLPA) The remaining
•96 patients underwent Genetic panel testing that included 52
peripheral neuropathy genes

In 35 patients (34 %) a genetic diagnosis was possible.
mutations were distributed among 15 different genes
•9 variants were classified as clearly pathogenic,
•19 as likely to be pathogenic and
•10 as being of unknown significance.
CONCLUSION:
In Hereditary Neuropathies- most profitable strategy for genetic
testing is NGS, after exclusion of PMP22 duplication/deletion
and possibly Sanger sequencing of GJB1

• It is a retrospective study indicating the role of WES in various
paediatric neurological disorders
• WES of 78 patients with unexplained Neuro developmental
Disorders who presented to the paediatric neurogenetics clinic at
Kennedy Krieger Institute b/w November 2011 and February 2014
was analysed

•pathogenic or likely pathogenic variants were diagnosed in 32
patients(41%)
•In rest of the 46 patients 32 had at least 1 variant of unknown
significance
Results of WES had several implications in Management
•Concerns regarding reproductive planning of parents in 27 patients
was addressed
•In 6 patients, WES results prompted further workup for systemic
involvement
•In 5 patients discontinued medications that was started on
presumptive diagnosis
•In 4 patients monitoring of the disease could be planned after
diagnosis
•In 3 patients therapeutic intervention was available

Diagnostic rates in various neurological disorders by NGS

ACMG Recommendations for Informed Consent for
Genome/Exome Sequencing
•Before initiating GS/ES, counselling should be performed by a
medical geneticist or an affiliated genetic counsellor and should
include written documentation of consent from the patient.
•Specific information regarding the clinical indication for GS/ES
need to be provided
•Counselling should include a discussion of the expected outcomes
of testing, the likelihood and type of incidental results that may be
generated

•Patients should be informed of the possibility of Incidental/secondary
findings that may have high clinical significance for which interventions
may exist to prevent or ameliorate disease severity.
•Patients should be informed to re-contact the referring physicians as
new knowledge is gained about the significance of particular results.
•Patients should be informed as to whether individually identifiable
results may be provided to databases, and they should be permitted to
opt out of such disclosure.
•Limitations of GS/ES in detecting trinucleotide repeat sequences,
translocation of gene, large deletions need to be addressed

ACMG Recommendations for Reporting of Incidental
Findings in Clinical Exome and Genome Sequencing
Primary Finding
This term is used to describe pathogenic alterations in a gene or
genes that are relevant to the diagnostic indication for which the
sequencing was ordered
Incidental Finding
Indicate the results of a deliberate search for pathogenic or likely
pathogenic alterations in genes that are not apparently relevant
to a diagnostic indication for which the sequencing test was
ordered

Clinical Utility of Incidental Findings
Reporting incidental findings in the following situations has
medical benefits
•Disorders where preventative measures are available
•Disorders where treatment is available and
•Disorders in which individuals with pathogenic mutations might
be asymptomatic for long periods of time.

ACMG recommends active search for the following specified types
of mutations in the specified genes based on clinical benefits listed
in these recommendations.

Limitations and Interpretation of Incidental Findings
•Analysis of genes for reporting the recommended incidental
findings, may not be technically equivalent to examining these
genes as a primary finding.
•Negative incidental findings is not equal to absence of
pathogenic variants in those genes
•Sequencing may not cover the genes examined for incidental
findings that would be filled in by Sanger sequencing or other
supplementary approaches if the gene were being evaluated for a
primary indication.

•Before reporting positive incidental findings laboratories need to
review available literature and databases at the time of the sequence
interpretation to ensure there is sufficient support for pathogenicity,
however presently there is no single database that has information
regarding all the possible variants of being pathogenic/non
pathogenic
•Clinician need to cautiously apply the information of incidental
findings for the patient in light of personal and family history, physical
examination, and other relevant findings
.

•Due to structural differences ,all portions of the Genome are not
sequenced equally and it is therefore possible that a mutation may
exist in a region of reduced coverage
•Variant interpretation
Whether a variant is pathogenic or not may not be answered by
existing database and it also depends on the technique used by
laboratories for variant assessment
Limitations of NGS

Conclusion
•Next-generation sequencing methodology allows for the rapid
and relatively inexpensive analysis of large amounts of DNA, up
to entire genomes.
•WES/WGS can broadly improve the diagnosis rate for
suspected neurogenetic disorders with clinically
heterogeneous phenotypes in both children and adults,
leading to benefits in patient care
•Clinical Exome /Genome sequencing should be utilized to
compliment, not supersede, a systematic comprehensive
patient evaluation. Physicians should receive proper training
on its appropriate use and interpretation of results.

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