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Whole Exome and Genome sequencing in Neurological disorders. The document discusses the techniques of next generation sequencing (NGS), including whole exome sequencing (WES) and whole genome sequencing (WGS). It provides examples of studies applying WES/WGS to diagnose neurological disorders, finding diagnostic variants in 25-60% of cases of leukoencephalopathy, limb-girdle muscular dystrophy, cerebellar ataxia, and more. The limitations and clinical applications of WES/WGS are also reviewed.

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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. Whole Exome and Genome sequencing in Neurological disorders Introduction
Whole Exome and Genome sequencing in Neurological disorders 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
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 Whole Exome and Genome sequencing in Neurological disorders 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 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 Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
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., $30,000 or more) Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
Chromosomal microarray analysis •It is a technique for detecting clinically significant structural changes, particularly deletions or duplications in the genome, copy number variation •As WES/WGS cannot detect sequence variation, CMA is used complementary in NGS to detect structural variation that Exome/Genome sequencing cannot Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques •First whole genome sequencing •It started in 1990 and completed in 2003 • Costed 3.8 billion$ •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 Exome and Genome sequencing in Neurological disorders
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 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 Whole Exome and Genome sequencing in Neurological disorders
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
Whole Exome and Genome sequencing in Neurological disorders Sample collection Technique of NGS
Whole Exome and Genome sequencing in Neurological disorders 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
Whole Exome and Genome sequencing in Neurological disorders 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
Whole Exome and Genome sequencing in Neurological disorders 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application •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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 . Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 Whole Exome and Genome sequencing in Neurological disorders Clinical application 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), Whole Exome and Genome sequencing in Neurological disorders Clinical application
Based on clinical indications, immunohistochemistry, candidate genetic testing was performed Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 ) Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
Cerebellar ataxias are a diverse collection of neurologic disorders Numerous genetic disorders have been associated with chronic progressive ataxia Whole Exome and Genome sequencing in Neurological disorders Clinical application 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
Whole Exome and Genome sequencing in Neurological disorders Clinical application 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
• 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%) Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
Whole Exome and Genome sequencing in Neurological disorders Clinical application •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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
pathogenic causative mutations was identified in 42%(21 of 50) patients by WGS Whole Exome and Genome sequencing in Neurological disorders Clinical application 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
• 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
Whole Exome and Genome sequencing in Neurological disorders Clinical application Diagnostic rates in various neurological disorders by NGS
Whole Exome and Genome sequencing in Neurological disorders Clinical application
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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
ACMG recommends active search for the following specified types of mutations in the specified genes based on clinical benefits listed in these recommendations. Whole Exome and Genome sequencing in Neurological disorders Clinical application
Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 . Whole Exome and Genome sequencing in Neurological disorders Clinical application
•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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
Thank you

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Ngs presentation

  • 1.
    Whole Exome andGenome sequencing in Neurological disorders
  • 2.
    •Introduction to Genetics •Definitions •Techniqueof NGS •Clinical Application •Limitations •Conclusion Over View Whole Exome and Genome sequencing in Neurological disorders
  • 3.
    •Human genome isa 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. Whole Exome and Genome sequencing in Neurological disorders Introduction
  • 4.
    Whole Exome andGenome sequencing in Neurological disorders 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
  • 5.
    Whole Exome andGenome sequencing in Neurological disorders Introduction Structure of a GENE
  • 6.
    DNA sequencing hasstarted with 2 dimensional Chromatography in 1970s that could read only 4-6 base pair length Whole Exome and Genome sequencing in Neurological disorders 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
  • 7.
    Generation 2-Automated sequencing In1987 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
  • 8.
    Methods for geneticdiagnostic 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 Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
  • 9.
    Multigene sequencing (Sangersequencing, 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., $30,000 or more) Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
  • 10.
    Chromosomal microarray analysis •Itis a technique for detecting clinically significant structural changes, particularly deletions or duplications in the genome, copy number variation •As WES/WGS cannot detect sequence variation, CMA is used complementary in NGS to detect structural variation that Exome/Genome sequencing cannot Whole Exome and Genome sequencing in Neurological disorders DNA sequencing Techniques
  • 11.
    Whole Exome andGenome sequencing in Neurological disorders DNA sequencing Techniques •First whole genome sequencing •It started in 1990 and completed in 2003 • Costed 3.8 billion$ •It used 1st and 2nd generation techniques HUMAN GENOME PROJECT
  • 12.
    DEFINITION Next-Generation Sequencing(NGS) Any processthat 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 Exome and Genome sequencing in Neurological disorders
  • 14.
    Whole-Genome Sequencing Determination ofthe 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 Whole Exome and Genome sequencing in Neurological disorders DEFINITION
  • 15.
    Whole Exome SequencingWhole 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 Whole Exome and Genome sequencing in Neurological disorders
  • 16.
    NGS experiments consistof 4 phases •Sample collection •Template generation • Amplification •Sequencing •Alignment and data analysis Whole Exome and Genome sequencing in Neurological disorders Technique of NGS
  • 17.
    Whole Exome andGenome sequencing in Neurological disorders Sample collection Technique of NGS
  • 18.
    Whole Exome andGenome sequencing in Neurological disorders 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
  • 19.
    Whole Exome andGenome sequencing in Neurological disorders 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
  • 20.
    Whole Exome andGenome sequencing in Neurological disorders 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
  • 21.
    Whole Exome andGenome 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
  • 22.
    Benefits of accuratemolecular 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
  • 23.
    Indications of NGS WholeExome and Genome sequencing in Neurological disorders Clinical application •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
  • 24.
    Neurological presentations ofgenetic 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 25.
    A total of191 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 . Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 26.
    •101 patients werediagnosed 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application 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
  • 27.
    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), Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 28.
    Based on clinicalindications, immunohistochemistry, candidate genetic testing was performed Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 29.
    Of the 154families 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 ) Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 30.
    In the remainingundiagnosed 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 31.
    Cerebellar ataxias area diverse collection of neurologic disorders Numerous genetic disorders have been associated with chronic progressive ataxia Whole Exome and Genome sequencing in Neurological disorders Clinical application 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
  • 32.
    Whole Exome andGenome sequencing in Neurological disorders Clinical application 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
  • 33.
    All patients withintractable 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 34.
    110 patients wereincluded, 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 35.
    Targeted next-generation sequencingpanels 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 36.
    Epilepsies have ahighly 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 37.
    • 33 caseswith 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%) Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 38.
    Conclusion: •Disease-specific gene panelallows 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 40.
    Whole Exome andGenome sequencing in Neurological disorders Clinical application •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
  • 41.
    •A pathogenic mutationexplaining 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 42.
    •Intellectual disability isa 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 43.
    pathogenic causative mutationswas identified in 42%(21 of 50) patients by WGS Whole Exome and Genome sequencing in Neurological disorders Clinical application 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
  • 44.
    Genetic heterogeneity in hereditaryperipheral neuropathy Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 45.
    •It was aretrospective 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 46.
    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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 47.
    • It isa 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 48.
    •pathogenic or likelypathogenic 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 49.
    Whole Exome andGenome sequencing in Neurological disorders Clinical application Diagnostic rates in various neurological disorders by NGS
  • 50.
    Whole Exome andGenome sequencing in Neurological disorders Clinical application
  • 51.
    ACMG Recommendations forInformed 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 52.
    •Patients should beinformed 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
  • 53.
    ACMG Recommendations forReporting 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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 54.
    Clinical Utility ofIncidental 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 55.
    ACMG recommends activesearch for the following specified types of mutations in the specified genes based on clinical benefits listed in these recommendations. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 57.
    Whole Exome andGenome sequencing in Neurological disorders Clinical application
  • 58.
    Limitations and Interpretationof 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 59.
    •Before reporting positiveincidental 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 . Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 60.
    •Due to structuraldifferences ,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 Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 61.
    Conclusion •Next-generation sequencing methodologyallows 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. Whole Exome and Genome sequencing in Neurological disorders Clinical application
  • 62.