Angelman Syndrome and UBE3A. Angelman Syndrome is a severe neurodevelopmental disorder characterized by intellectual disability, typical facial features and a usually happy demeanor. Patients with Angelman Syndrome usually do not acquire active speech and often show a characteristic, atactic gait. Also, patients with Angelman Syndrome have a characteristic EEG pattern and many children have seizures. Angelman Syndrome is a genetic disorder due to loss of function of UBE3A, a ubiquitin ligase expressed in the CNS. Ubiquitin ligases are the bin collectors of the cell. By attaching ubiquitin to proteins, proteins are labelled for cellular degradation. How a malfunction of a cellular garbage truck causes such a complex neurodevelopmental disorder is poorly understood. A recent study, however, points out an important role for interneurons…. Continue reading
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FAME – when phenotypes cross over but chromosomes don’t
Crompton and colleagues recently published the clinical and genetic description of a large family with Familial Adult Myoclonic Epilepsy (FAME). This phenotype is particularly interesting since it provides some insight into how neurologists conceptualize twitches and jerks. It is also a good example that large families do not necessarily result in a narrow linkage region, particularly when centromeric regions are involved.
What is myoclonus? Despite usually mentioned in the context of epilepsy, most people are inherently familiar with myoclonus. Most of us “twitch” when we fall asleep and sometimes experience this twitch as part of a dream. These episodes are entirely normal and are called hypnic jerks, but they give people a good idea of what a sudden, brief, shocklike, involuntary movement caused by muscular contraction or inhibition would feel like. Myoclonus in the setting of epilepsy is usually mentioned as part of a Juvenile Myoclonic Epilepsy (JME) or Progressive Myoclonus Epilepsy (PME). Please note that both epilepsies use different endings to describe the twitch (“-us” vs. “–ic”). This is mainly convention. Basically, myoclonus is a brief shock-like twitch, which can affect almost every part of the body and can be due to dysfunctions in various regions in the Central Nervous System.
The neuroanatomy of twitching. A motor command from the cerebral cortex has to pass through several steps prior to execution. For example, the simple command of tapping a finger on the table surface is prepared by the cortex through several loops before being sent down your spine. Accordingly, myoclonus can arise from different parts in the brain. (1) The cortical myoclonus is due to a purely cortical source and can be seen in many forms of symptomatic myoclonus. (2) The cortico-subcortical myoclonus is due to feedback from the cortex to other brain areas. This is the myoclonus we see in patients with JME. Both variants may be seen on EEG since the cortex is involved. (3) The subcortical-supraspinal myoclonus is generated in the brain stem or below and is responsible for phenomena such as hyperekplexia or startle disease. Some forms of hyperekplexia, literally “exaggerated surprise”, are due to mutations in genes involved in glycinergic transmission and can be found in some isolated communities such as the Jumping Frenchmen of Maine. (4) Finally, there is also spinal and peripheral myoclonus.
FAME – epilepsy or movement disorder? Familial Adult Myoclonic Epilepsy (FAME) is an enigmatic familial disorder with the triad of myoclonus, tremor and seizures. Several families have been described and two loci on 8q23.3-8q24.11 and 2p11.1-q212.2 for FAME have been established. The underlying genes are still unknown. Crompton and colleagues no describe a large six-generation family with FAME in Australia/New Zealand. The familial disease usually starts with tremor in early adulthood in the affected family members, even though a wide range of age of onset is observed. Interestingly, only a quarter of all affected family members had seizures, which is in contrast to previous studies. Therefore, FAME may actually be better characterized as a movement disorder with concomitant seizures rather than a familial epilepsy syndrome. The authors also point out the difficulties distinguishing FAME from the much more common essential tremor (ET). In particular, the well-described response to β-blockers seen in patients with ET can also be observed in some family members.
The genetics of FAME. Crossovers during meiosis usually lead to a progressive narrowing of the linkage interval in familial disorders. However, the lack of crossover events leads to very large linkage intervals even in very extended families. The family described by Crompton et al. links to the pericentromeric region of chromosome 2. Pericentromeric regions usually have a low frequency of crossover events, and this phenomenon has also delayed the identification of other familial epilepsies such as Benign Familial Infantile Seizures with mutations in PRRT2. The linkage region contains almost 100 genes and Figure 1 shows the “candidate gene landscape” in this region. While some genes clearly classify as top candidate genes, the majority of the genes in this region are unknown in the context of epilepsy. Therefore, identification of the FAME gene will be exciting and provide us with novel insight on how genetic alterations may produce combined neurological phenotypes.
Be literate when the exome goes clinical
Exomes on Twitter. Two different trains of thoughts eventually prompted me to write this post. First, a report of a father identifying the mutation responsible for his son’s disease pretty much dominated the exome-related twittersphere. In Hunting down my son’s killer, Matt Might describes his family’s journey that finally led to the identification of the gene coding for N-Glycanase 1 as the cause of his son’s disease, West Syndrome with associated features such as liver problems. The exome sequencing that finally led to the discovery was part of a larger program on identifying the genetic basis of unknown, putatively genetic disorders reported in a paper by Anna Need and colleagues, which is available through open access. This paper is an interesting proof-of-principle study that exome sequencing is ready for prime time. Need and colleagues suggest exome sequencing can find causal mutations in up to 50% of patients. By the way, a gene also that turned up again was SCN2A in a patient with severe intellectual disability, developmental delay, infantile spasms, hypotonia and minor dysmorphisms. This represents a novel SCN2A-related phenotype, expanding the spectrum to severe epileptic encephalopathies.
The exome consult. My second experience last week was my first “exome consult”. A colleague asked me to look at a gene list of a patient to see whether any of the genes identified (there were 300+ genes) might be related to the patient’s epilepsy phenotype. Since I wasn’t sure how to best handle this, I tried to run an automated PubMed search for combination of 20 search terms with a small R script I wrote. Nothing really convincing came up except the realisation that this will be an issue that we will be increasingly faced in the future: working our way through exome dataset after the first “flush” of data analysis did not reveal convincing results. Two terms that came to my mind were bioinformatic literacy as something that we need to improve and Program or be Programmed, a book by Douglas Rushkoff on the “Ten commands of the Digital Age”. In his book, he basically points out that in the future, understanding rather than simply using IT will be crucial.
The cost of interpretation is rising. The Genome Center in Nijmegen suggests on their homepage that by the year 2020, whole-genome sequencing will be a standard tool in medical research. What this webpage does not say is that by 2020, 95% of the effort will not go into the technical aspects of data generation, but into data interpretation. For biotechnology, interpretation will be the largest marketing sector.
So, what about epilepsy? “50% of cases to be identified” sounds good for any grant proposal that I would write, but this might be a clear overestimate. Need and colleagues used a highly selected patient population and even in the variants they identified, causality is sometimes difficult to assess. We are maybe much further away from clinical exome sequencing in the epilepsies than we would like to admit. The only reference point we have for seizure disorders to date is large datasets for patients with autism and intellectual disability. While some genes with overlapping phenotypes can be identified, we would virtually be drowning in exome data without being capable of making sense of this.
10,000 exomes now. I would like to predict that after having identified some low-hanging fruits with monogenic disorders, 10,000 or more “epilepsy exomes” would have to be collected before making significant progress. It is, therefore, crucial not to be tempted by wishful thinking that particular epilepsy subtypes necessarily have to be monogenic, as in the case of epileptic encephalopathies or other severe epilepsies. Much of the genetic architecture of the epilepsies might be more complex than anticipated, requiring larger cohorts and unanticipated perseverance.
The Marrakesh diaries – DNA colonialism
A 21st century gold rush. Collections of biosamples, referred to as biobanks, are sometimes referred to as the ‘gold of the 21st century‘, as these collections will provide the key for translating the findings of biomedical research into patient treatments. The upcoming revolution of personalized health can only happen if well-curated patient samples for DNA, tissues and other biomaterials are available. In many European countries, large government-funded initiatives are on the way to build these collections. So far, so good.
DNA colonialism. But what does this have to do with colonialism? The phrase of DNA colonialism has a dual origin and was pretty much invented in parallel in a discussion I had with researches in Israel and Morocco. Given that this idea came up twice independently within a few weeks, it prompted me to put this together as a blog entry. DNA colonialism refers to the phenomenon that researchers from “developed” countries obtain valuable biosamples in “developing” countries for their research. Collaborations with emerging countries are becoming increasingly important given the particular genetic architecture in these countries, which lends itself to gene discovery. Often collaborating researchers in the emerging country are only involved on a very basic level and are sometimes not even involved in the final publication of the data. This phenomenon is frequently observed in the literature when the author list of novel gene findings in consanguineous families do not include researchers from the respective emerging country.
DNA mining leaves little behind other than empty mines. Within these bilateral collaborations, the genetic architecture of the “developing” countries is mined by Western researchers, which is sometimes interpreted as a modern form of colonialism. While there is little doubt that the findings originating from this research are important, there is little benefit for the emerging country. Examples where the gene findings in families are translated into screening programs are rare and -to my knowlegde- only exists for Bedouin population in the Southern part of Israel. Treatment options based on these findings are even rarer. Instances, in which a partnership between a “developing” and “developed” country has resulted in the creation of infrastructure on site are few.
New rules for “DNA trading”. What has to be done to avoid DNA colonialism and what would constitute a fair trade agreement to enable a productive partnership rather than an exploitation of the genetic architecture? Naturally, there is not a single definite solution for this issue, but at least two points may be raised in this context.
Biosamples are becoming more valuable. First, the relative value of biosamples in relation to genetic technologies is increasing. The price for Next Generation Sequencing technologies is constantly dropping and samples can be analyzed at much lower costs. This will naturally help the relationship between both partners as the effort to obtain sample is increasingly valued. Also, there is an increasing awareness regarding the IRB-related issues surrounding biosamples. While many researchers still feel that they lose the control over a given biosample once the sample leaves the country, the entire field is getting increasingly sensitized to these issues. Modern material transfer agreements might include well laid-out plans for what happens with samples once they cross international borders.
Redistribution, fostering intrinsic motivation. Secondly, research environments in developing countries would need to provide a commitment towards generating a sustainable infrastructure in emerging countries. Despite the naive impression that building good research environments is not possible in countries outside the Western sphere, there are examples that suggest otherwise. For example, the Kanaan lab in Bethlehem, Palestine, represents one of the of the premier labs worldwide for the research of genetic hearing loss and Dr. Kanaan has a strong commitment to establishing methods and technologies on site. As in many other instances, lack for funding for R&D is not a matter of resources, but of distribution. The question of to what extent pure external incentives such as large amounts of funding might help resolve these issues is uncertain, and one of the key challenges would be to foster intrinsic motivation for these issues in young researchers.
Implementing some of these issues might help researchers in emerging countries establish long-term plans to generate on-site know-how and infrastructure in order to fully participate as equal partners in international research networks. Eventually, the hunt for epilepsy genes does only start with the identification of these variants. If we ever have the hope that genetic findings in the epilepsies will impact on patient care and treatment, we as the EuroEPINOMICS consortium should strongly motivate our collaborative partners in emerging countries to be more than mere sample providers.
Next Generation Sequencing as a diagnostic tool in the epilepsy clinic
Remember Guthrie cards and the heel stick for newborn screening? It will be a thing of the past in 10 years replaced by methods performed through Next Generation Sequencing (NGS). NHGRI and NICHD have already committed to a $25M program for Next Generation Sequencing in Newborn Screening and first reports appear describing the value of exome sequencing in solving undiagnosed cases. However, these reports all leave clinicians working in the epilepsy clinic scratching their heads – this all sounds very good, but what can you offer your patients already, not just in 2-3 years?
265 genes at once. A team led by the EuroEPINOMICS researchers Johannes Lemke and Saskia Biskup has now evaluated the feasibility of targeted Next Generation Sequencing of a panel of epilepsy genes and the results published in Epilepsia last week are quite impressive. With their panel of 265 genes, they identified mutations in 16/33 patients with unclassified, presumably genetic epilepsy. While the overall yield of this candidate panel is probably lower than the impressive 50% in their pioneer study, these results clearly show that the general workflow in the epilepsy clinic is ready to shift from candidate gene screening to Next Gen panel analysis.
New and old genes identified. The list of genes identified in their screening is a mixed bag of epilepsy genes, many of which were identified in syndromes with a high degree of clinical suspicion including mutations in SCN1A, SCN2A and KCNQ3. Interestingly, some unlikely candidates also popped up. One patient with a clinical picture of Dravet Syndrome (DS) had a mutation in TPP1, the gene causative for Neuronal Ceroid Lipofuscinosis Type 2. This unexpected finding highlights another important “side-effect” of NGS: we will probably discover many unusual phenotypes for known disorders.
You wouldn’t think so, but panels are sometimes more thorough. Lemke and coworkers identify mutations in SCN1A in three patients with DS. This alone would not be all that remarkable. However, these three patients were previously reported to be negative for SCN1A by Sanger sequencing. This phenomenon is not new. In addition to identifying GABRA1 in SCN1A-negative DS, Mefford and colleagues also identified a mutation in SCN1A by exome in a patient with DS that was missed by conventional sequencing. While it is difficult to compare exome and conventional sequencing, these two anectodes at least suggest that NGS is not fairing any worse than conventional methods.
Targeted sequencing vs. exome. In the upcoming 12-24 months, we expect an intense debate on whether targeted sequencing is actually necessary or whether you could directly apply diagnostic exome sequencing. Targeted technologies – for now – have the advantage of the higher coverage, i.e. the eventual quality and completeness of candidate gene sequences higher than in exome studies. However, the field is evolving and the next, better technology might already be around the corner.
The Marrakesh diaries – The paradox of autosomal recessive common epilepsies
When in epilepsy genetics, choose sides. When you (a young, motivated researcher) start working on epilepsy genetics, you have to make a basic decision. Option 1 is the decision to work on the genetics of rare epilepsies. Rare epilepsies, either monogenic families or epileptic encephalopathies are expected to have a strong genetic contribution. In the ideal situation, a gene can be identified if the family is sufficiently large or if you happen to pick the right phenotype. Option 2 involves genetic research in common epilepsies. In this case, you can claim to be working on the seizure disorders which represent more than 90% of patients. Identification of genetic risk factors in this group of patients undoubetedly has strong implications, be it the identification of risk factors for drug response, side effects or novel risk variants, which help you understand the underlying biology. However, few of these risk factors are known and large sample sizes are needed to create a sufficiently powered study. Interestingly, the distinction between these two options pretty much represent the differences between RES and CoGIE.
Why not Option 3? Whichever option you chose, it has apparent downsides. You either work on very rare diseases or exhaust yourselve with more genetic complexity than you can handle. There is, however, Option 3. And Option 3 is what led part of the EuroEPINOMICS blogging team (namely Ingo, Johanna and Sarah) to Marrakesh. Option 3 involves genetic studies in familial forms of common epilepsies, particularly autosomal recessive variants of common seizure disorders.
The paradox. For disorders such as Juvenile Myoclonic Epilepsy (JME), we expect a complex genetic architecture on the population level with a wide range of genetic risk factors, a high degree of locus heterogeneity and phenotypic pleiotropy of risk variants. However, families have been reported, in which such common forms of seizure disorders are inherited in an autosomal recessive manner. For Juvenile Myoclonic Epilepsy, for example, several families have been reported with recessive inheritance. Furthermore, Salzmann and colleagues recently identified a mutation in the gene coding for Carboxypeptidase A6 in a recessive family with Temporal Lobe Epilepsy and Febrile Seizures. These families provide the ideal opportunity to identify genetic risk factors with strong effect, which would help us understand basic mechanism. The downside, however, is that these families are extremely rare. This, again, is where Marrakesh comes into play.
Genetic research in populations with a high degree of consanguinity. In several populations around the world, consanguineous marriages are frequent. Marriage within the family sometimes represents an important social factor. This constellation is seen in many Arab populations. Autosomal recessive disorders are relatively frequent in these populations and represent an important source of morbidity. Interestingly, highly consanguineous population do not have a significant increase in the frequency of common disorders, suggesting that the effect of recessive mutations occurs in a black-and-white fashion, i.e. these mutations cause severe monogenic disorders when present in a recessive state, but do not contribute strongly when heterozygous. Highly consanguineous population, therefore, lend themselves for gene discovery in recessive disorders. The frequency of these disorders is higher and identification of the underlying gene is highly important to the families and population for screening.
What is recessive JME? You might argue that recessive Juvenile Myoclonic Epilepsy does not exist and probably represents a form of Progressive Myoclonus Epilepsy (PME) which either present in a mild form or is not properly diagnosed. While it is difficult to argue against the presence of an atypical phenotype in the absence of genetic data, a long non-progressive course of a mild myoclonic epilepsy without ataxia or additional features is difficult to reconsile with PME. And PME genes are notoriously absent in cohorts of patients with JME. These disorders are simply distinct. Therefore, recessive JME holds the great promise to identify novel recessive genes for IGE/GGE that will undoubtely unravel novel mechanisms. From the top of my head, I would not know a single gene that, when absent in a homozygous state, might cause JME and the mechanisms to be identified hold great promise. The Morocco trip (supported by the International Bureau of the BMBF) has allowed us to already include some families into the EuroEPINOMICS project with more families to come.