Ngs presentation

Whole Exome and Genome sequencing in Neurological disorders

•Introduction to Genetics•Definitions•Technique of NGS•Clinical Application•Limitations•Conclusion

Over View


•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

GeneA 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.

Whole Exome and Genome sequencing in Neurological disorders Introduction


Each Gene consists of Regulatory component and Reading Frame•Regulator region includes -Enhancer/silencer and -Promoter/TerminatorReading 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

Whole Exome and Genome sequencing in Neurological disorders 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 sequencingIn 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

Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques

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 interestAccordingly there are several applications of NGS

Sequencing of entire Genome /Exome is primarily used in clinics


Whole-Genome SequencingDetermination 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 SequencingDetermination 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

Whole Exome and Genome sequencing in Neurological disorders 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

Whole Exome and Genome sequencing in Neurological disorders 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

Whole Exome and Genome sequencing in Neurological disorders 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

Whole Exome and Genome sequencing in Neurological disorders 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 cognitiveDysfunction 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 mutationsCONCLUSION: 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

Clinical application Whole Exome and Genome sequencing in Neurological disorders

ACMG Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing

Primary FindingThis term is used to describe pathogenic alterations in a gene or genes that are relevant to the diagnostic indication for which the sequencing was orderedIncidental FindingIndicate 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

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•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.


Thank you

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