J Neurol (2014) 261:1244–1246 DOI 10.1007/s00415-014-7372-1
JOURNAL CLUB
Applications of next-generation whole exome sequencing Katharine E. Harding • Neil P. Robertson
Published online: 18 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Since the publication of the first human genome in 2001, significant advances in understanding the genetic aetiology of neurological diseases have been made. However, genetic heterogeneity, relative rarity of clinical presentation, and high cost of current genetic sequencing methods have limited identification of causative mutations in many other patients. It is becoming apparent that a range of mutations may cause similar clinical phenotypes, which means that consecutive Sanger sequencing of individual genes is time consuming and very expensive, and still may not provide a final answer. Whole exome sequencing (WES) is a new genetic sequencing technique which is able to circumvent some of these difficulties. Only the exons are sequenced, i.e. only the genetic regions which will be translated into proteins and thus be expected to have a functional or structural effect. Each exon is sequenced multiple times, allowing the sequencing to be checked within each subject and thus increasing confidence that the final sequence is accurate. This technique is, therefore, also known as massively parallel sequencing. It can be applied to all exons, or can be used to focus on a particular region of the genome such as a section of a chromosome which has previously been identified as being of interest, or it can be used to sequence a set of genes which are not necessarily genomic neighbours. WES has a number of advantages as compared to Sanger sequencing. First, it is not essential to know in advance which gene needs to be sequenced, and allows screening of genes which may not have been suspected to be associated K. E. Harding N. P. Robertson (&) Department of Neurology, Institute of Psychological Medicine and Clinical Neuroscience, Cardiff University, Cardiff, UK e-mail: [email protected]
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with the disease in question. Second, the incorporation of within-patient checks by multiple sequencing of exons means that mutations which had been overlooked by Sanger sequencing may be detected. Finally, it is significantly cheaper than Sanger sequencing and WES may now be performed for a similar cost to Sanger sequencing of one or two genes. However, there are also some drawbacks: some areas of the genome are not covered as well as others by these techniques. There is also the possibility of detecting mutations that are known to cause other genetic diseases which may be unrelated to the current clinical presentation, for example BRCA1, and so there are additional ethical considerations to be taken into account. In this month’s journal club, we have reviewed four papers that use next-generation sequencing techniques in a range of different neurological problems. The first paper examines DOORS, a rare disease, using a multi-centre collaborative approach to identify patients and illustrates the importance of clinical input to obtain an accurate phenotype. The second paper focuses on Dravet syndrome, and shows that it may be associated with more than one genetic mutation, which has implications for subsequent clinical management of patients. The third paper applies WES techniques to patients with suspected inherited peripheral neuropathies and where a genetic diagnosis had not been found—a common clinical scenario. Finally, we discuss another paper where careful phenotyping identified a small number of similar patients, and sequencing revealed a likely mitochondrial genetic mutation, which again may have clinical implications for those patients. Together these papers illustrate how WES may be used in clinical neurology to refine diagnosis and improve management, although we are still in the very early stages of understanding genetic contribution to neurological disease.
J Neurol (2014) 261:1244–1246
The genetic basis of DOORS syndrome: an exomesequencing study DOORS syndrome (deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures) is a very rare condition, where previous genetic techniques had been unsuccessful in identifying causative mutations, in part due to small numbers and phenotypic variability. This international multi-centre study identified 30 families and used WES in 17, which revealed recessive mutations in TBC1D24 in 7 families. This was then sequenced using Sanger sequencing in 9 further families as a validation cohort, and TBC1D24 mutations were identified in two more families. Comment and conclusions. This study demonstrates the value of multi-centre collaboration, without which it would not have been possible to establish genetic mutations associated with DOORS syndrome. Furthermore, it illustrates the importance of clinical phenotyping to accurately define presentation of disease and ensure that a clinically heterogenous population is being studied. TBC1D24 is curious as it has been previously associated with several other epilepsy syndromes, each phenotypically quite different, and thus far it has been unusual for a recessive mutation to cause such a wide range of phenotypes. As yet, this study does not suggest a mechanism that might underlie DOORS syndrome, and functional studies will be important in this respect. Campeau P et al (2014) Lancet Neurol 13: 44–58.
GABRA1 and STXBP1: novel genetic causes of Dravet syndrome Dravet syndrome is a rare inherited form of infantile-onset epileptic encephalopathy with a poor prognosis. Around 75 % of cases are caused by a mutation in the sodium channel a-1 subunit (SCNA1), and a small number of cases with mutations in other genes have been identified, but the cause remains unknown in around a fifth of cases. This paper used WES in 13 Dravet SCN1A-negative cases, diagnosed on clinical grounds. Targeted sequencing of 15 candidate genes identified from this stage was then carried out in a validation cohort of 67 Dravet SCN1A-negative cases. In 3/15 patients, previously overlooked mutations in SCN1A were identified, suggesting that more than 75 % of cases may be caused by SCN1A. Further pathogenic mutations were identified in GABRA1 and STXBP1, both of which are known to cause other epileptic syndromes. Finally, functional studies were carried out to understand the GABRA1 mutation, by inserting the mutated receptor into oocytes, stimulating with GABA and measuring the
1245
amplitude of the electrochemical response. The response of the mutated receptor was significantly diminished compared with wild type. Comment and conclusions. In addition to showing that mutations in SCN1A are responsible for more cases of Dravet syndrome than had previously been recognised, this study has widened the clinical phenotype associated with mutations in GABRA1 and STXBP1, and also suggested how GABRA1 mutations may affect electrochemical responses in a cell. Although traditional sequencing techniques have significantly advanced our understanding of Dravet syndrome which has allowed specific treatments to have been developed, this study demonstrates how WES can be used to enhance our understanding of genetic epilepsy syndromes and suggest possible mechanisms underlying disease. Carvill G et al (2014) Neurology 82: 1245–1253.
Application of whole exome sequencing in undiagnosed inherited polyneuropathies Inherited polyneuropathies are notoriously difficult to diagnose, due to extensive genetic heterogeneity. This study gathered 24 cases from 15 kindreds where traditional sequencing had not identified causative mutations, and performed WES of 74 candidate genes, based on a recent review of all genes previously associated with polyneuropathies. In five kindreds, known pathogenic mutations were identified, which had not been picked up using traditional Sanger sequencing. In three kindreds, novel variants were identified in known neuropathyassociated genes—these segregated with clinical phenotype and were not recorded in any control database (over 10,000 genomes), making them very likely to be pathogenic. In seven kindreds, no causal mutation was found although multiple novel variants in non-neuropathy-associated genes were identified which will require further evaluation. Comment and conclusions. This study again highlights how even known pathogenic mutations may be overlooked by traditional Sanger sequencing techniques. As the cost of WES drops, this type of targeted approach to genetically heterogenous conditions will become more useful and accessible to clinical practice. Furthermore, it is likely to be significantly quicker and more cost effective that serial sequencing of candidate genes, which is both costly and time consuming. This is certainly a promising development and we look forward to future advances. Klein C et al (2014) J Neurol Neurosurg Psychiatry. Advance online publication, 6 Mar 2014. doi: 10.1136/ jnnp-2013-306740.
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Delineation of C12orf65-related phenotypes: a genotype–phenotype relationship In this study, two consanguineous families with multiple affected members with optic atrophy, pes cavus and spastic paraparesis were investigated. Following initial screening to exclude genetic regions shared with unaffected siblings, a large region shared by all affected patients in the first family was identified, and exome-sequencing techniques applied to this region. A deletion in C12orf65 was identified, which would result in a premature stop codon and truncated protein. A mutation in C12orf65 was also identified in the second family. In both families the mutation segregated perfectly with affected status. C12orf65 is an auxiliary factor in mitochondrial DNA translation
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J Neurol (2014) 261:1244–1246
machinery and, therefore, is likely to affect mitochondrial function. Comment and conclusions. This study is a well-designed and technically high-quality study of a small number of affected patients. Although this particular presentation is rare and unlikely to be encountered by the clinician, collectively patients with unidentified but suspected genetic neurological diseases are not uncommon, and this paper illustrates how new genetic techniques may be applied in such situations. Finding a mutation which is likely to be pathogenic is useful for counselling families although specific treatments may not yet be available. Spiegel R et al (2014) Eur J Hum Genet. Advance online publication, 15 Jan 2014. doi: 10.1038/ejhg.2013.284.
JOURNAL CLUB
Applications of next-generation whole exome sequencing Katharine E. Harding • Neil P. Robertson
Published online: 18 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Since the publication of the first human genome in 2001, significant advances in understanding the genetic aetiology of neurological diseases have been made. However, genetic heterogeneity, relative rarity of clinical presentation, and high cost of current genetic sequencing methods have limited identification of causative mutations in many other patients. It is becoming apparent that a range of mutations may cause similar clinical phenotypes, which means that consecutive Sanger sequencing of individual genes is time consuming and very expensive, and still may not provide a final answer. Whole exome sequencing (WES) is a new genetic sequencing technique which is able to circumvent some of these difficulties. Only the exons are sequenced, i.e. only the genetic regions which will be translated into proteins and thus be expected to have a functional or structural effect. Each exon is sequenced multiple times, allowing the sequencing to be checked within each subject and thus increasing confidence that the final sequence is accurate. This technique is, therefore, also known as massively parallel sequencing. It can be applied to all exons, or can be used to focus on a particular region of the genome such as a section of a chromosome which has previously been identified as being of interest, or it can be used to sequence a set of genes which are not necessarily genomic neighbours. WES has a number of advantages as compared to Sanger sequencing. First, it is not essential to know in advance which gene needs to be sequenced, and allows screening of genes which may not have been suspected to be associated K. E. Harding N. P. Robertson (&) Department of Neurology, Institute of Psychological Medicine and Clinical Neuroscience, Cardiff University, Cardiff, UK e-mail: [email protected]
123
with the disease in question. Second, the incorporation of within-patient checks by multiple sequencing of exons means that mutations which had been overlooked by Sanger sequencing may be detected. Finally, it is significantly cheaper than Sanger sequencing and WES may now be performed for a similar cost to Sanger sequencing of one or two genes. However, there are also some drawbacks: some areas of the genome are not covered as well as others by these techniques. There is also the possibility of detecting mutations that are known to cause other genetic diseases which may be unrelated to the current clinical presentation, for example BRCA1, and so there are additional ethical considerations to be taken into account. In this month’s journal club, we have reviewed four papers that use next-generation sequencing techniques in a range of different neurological problems. The first paper examines DOORS, a rare disease, using a multi-centre collaborative approach to identify patients and illustrates the importance of clinical input to obtain an accurate phenotype. The second paper focuses on Dravet syndrome, and shows that it may be associated with more than one genetic mutation, which has implications for subsequent clinical management of patients. The third paper applies WES techniques to patients with suspected inherited peripheral neuropathies and where a genetic diagnosis had not been found—a common clinical scenario. Finally, we discuss another paper where careful phenotyping identified a small number of similar patients, and sequencing revealed a likely mitochondrial genetic mutation, which again may have clinical implications for those patients. Together these papers illustrate how WES may be used in clinical neurology to refine diagnosis and improve management, although we are still in the very early stages of understanding genetic contribution to neurological disease.
J Neurol (2014) 261:1244–1246
The genetic basis of DOORS syndrome: an exomesequencing study DOORS syndrome (deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures) is a very rare condition, where previous genetic techniques had been unsuccessful in identifying causative mutations, in part due to small numbers and phenotypic variability. This international multi-centre study identified 30 families and used WES in 17, which revealed recessive mutations in TBC1D24 in 7 families. This was then sequenced using Sanger sequencing in 9 further families as a validation cohort, and TBC1D24 mutations were identified in two more families. Comment and conclusions. This study demonstrates the value of multi-centre collaboration, without which it would not have been possible to establish genetic mutations associated with DOORS syndrome. Furthermore, it illustrates the importance of clinical phenotyping to accurately define presentation of disease and ensure that a clinically heterogenous population is being studied. TBC1D24 is curious as it has been previously associated with several other epilepsy syndromes, each phenotypically quite different, and thus far it has been unusual for a recessive mutation to cause such a wide range of phenotypes. As yet, this study does not suggest a mechanism that might underlie DOORS syndrome, and functional studies will be important in this respect. Campeau P et al (2014) Lancet Neurol 13: 44–58.
GABRA1 and STXBP1: novel genetic causes of Dravet syndrome Dravet syndrome is a rare inherited form of infantile-onset epileptic encephalopathy with a poor prognosis. Around 75 % of cases are caused by a mutation in the sodium channel a-1 subunit (SCNA1), and a small number of cases with mutations in other genes have been identified, but the cause remains unknown in around a fifth of cases. This paper used WES in 13 Dravet SCN1A-negative cases, diagnosed on clinical grounds. Targeted sequencing of 15 candidate genes identified from this stage was then carried out in a validation cohort of 67 Dravet SCN1A-negative cases. In 3/15 patients, previously overlooked mutations in SCN1A were identified, suggesting that more than 75 % of cases may be caused by SCN1A. Further pathogenic mutations were identified in GABRA1 and STXBP1, both of which are known to cause other epileptic syndromes. Finally, functional studies were carried out to understand the GABRA1 mutation, by inserting the mutated receptor into oocytes, stimulating with GABA and measuring the
1245
amplitude of the electrochemical response. The response of the mutated receptor was significantly diminished compared with wild type. Comment and conclusions. In addition to showing that mutations in SCN1A are responsible for more cases of Dravet syndrome than had previously been recognised, this study has widened the clinical phenotype associated with mutations in GABRA1 and STXBP1, and also suggested how GABRA1 mutations may affect electrochemical responses in a cell. Although traditional sequencing techniques have significantly advanced our understanding of Dravet syndrome which has allowed specific treatments to have been developed, this study demonstrates how WES can be used to enhance our understanding of genetic epilepsy syndromes and suggest possible mechanisms underlying disease. Carvill G et al (2014) Neurology 82: 1245–1253.
Application of whole exome sequencing in undiagnosed inherited polyneuropathies Inherited polyneuropathies are notoriously difficult to diagnose, due to extensive genetic heterogeneity. This study gathered 24 cases from 15 kindreds where traditional sequencing had not identified causative mutations, and performed WES of 74 candidate genes, based on a recent review of all genes previously associated with polyneuropathies. In five kindreds, known pathogenic mutations were identified, which had not been picked up using traditional Sanger sequencing. In three kindreds, novel variants were identified in known neuropathyassociated genes—these segregated with clinical phenotype and were not recorded in any control database (over 10,000 genomes), making them very likely to be pathogenic. In seven kindreds, no causal mutation was found although multiple novel variants in non-neuropathy-associated genes were identified which will require further evaluation. Comment and conclusions. This study again highlights how even known pathogenic mutations may be overlooked by traditional Sanger sequencing techniques. As the cost of WES drops, this type of targeted approach to genetically heterogenous conditions will become more useful and accessible to clinical practice. Furthermore, it is likely to be significantly quicker and more cost effective that serial sequencing of candidate genes, which is both costly and time consuming. This is certainly a promising development and we look forward to future advances. Klein C et al (2014) J Neurol Neurosurg Psychiatry. Advance online publication, 6 Mar 2014. doi: 10.1136/ jnnp-2013-306740.
123
1246
Delineation of C12orf65-related phenotypes: a genotype–phenotype relationship In this study, two consanguineous families with multiple affected members with optic atrophy, pes cavus and spastic paraparesis were investigated. Following initial screening to exclude genetic regions shared with unaffected siblings, a large region shared by all affected patients in the first family was identified, and exome-sequencing techniques applied to this region. A deletion in C12orf65 was identified, which would result in a premature stop codon and truncated protein. A mutation in C12orf65 was also identified in the second family. In both families the mutation segregated perfectly with affected status. C12orf65 is an auxiliary factor in mitochondrial DNA translation
123
J Neurol (2014) 261:1244–1246
machinery and, therefore, is likely to affect mitochondrial function. Comment and conclusions. This study is a well-designed and technically high-quality study of a small number of affected patients. Although this particular presentation is rare and unlikely to be encountered by the clinician, collectively patients with unidentified but suspected genetic neurological diseases are not uncommon, and this paper illustrates how new genetic techniques may be applied in such situations. Finding a mutation which is likely to be pathogenic is useful for counselling families although specific treatments may not yet be available. Spiegel R et al (2014) Eur J Hum Genet. Advance online publication, 15 Jan 2014. doi: 10.1038/ejhg.2013.284.
