Of steel mills and exomes – the Luxembourg data analysis meeting

In the shade of the furnaces. The EuroEPINOMICS consortium met for the first data analysis meeting at the Luxembourg Center for System Biomedicine (LCSB) from July 5-7th, 2012. What was intended to be a small, private meeting on data analysis eventually turned into a medium-size consortium meeting with lively, sometimes revealing discussions. Belval is a campus in transition, a large steel mill that is currently transformed into the new campus of the University of Luxembourg. The LCSB people are the “first kids on the block”. The atmosphere of Belval is a mixture of industrial romance and pioneer spirit, the ideal backdrop for re-considering our current approaches to deciphering the genetics of the epilepsies. Continue reading

The meeting of the 1000 exomes

1000 exomes. The EuroEPINOMICS consortium will host its first data analysis meeting at the Luxembourg Centre for System Biomedicine from July 5th to July 7th, 2012, to discuss the results from the high-throughput genomic platforms in the CoGIE, RES, EpiGENet and Epiglia consortia. We will present the first results of the four EuroEPINOMICS programs generated by high-throughput genomic technologies and discuss the overall direction of genetic analysis for the next years, which should soon encompass the proverbial 1000 exomes. In preparation, let’s revisit high-throughput epilepsy genomics. Continue reading

Exome sequencing corrects diagnosis in autosomal recessive disorders

The amazing powers of exome sequencing – a disclaimer. We have recently blogged frequently on the power of exome sequencing in monogenic disorders. Dixon-Salazar now describe in “Exome Sequencing Can Improve Diagnosis and Alter Patient Management” the usefulness of exome sequencing in disease identification in autosomal recessive disorders. Their overall yield is a novel gene discovery in 22/118 probands and a different diagnosis than the initial in 10/118 patients. While title and abstract suggest that exome sequencing is a cure-all improving patient diagnosis and altering patient management, it should be pointed out that this manuscript exclusively deals with autosomal recessive disorders. Only two novel genes out of 20 are described, leaving the reader with little chance to investigate their claim. Many of their families were selected from countries with a high consanguinity including Morocco, where state-of-the-art diagnostic facilities are difficult to access for some patients. The only change in patient management resulting from the altered diagnosis was stopping supplementary Vitamin E in a family with a SPG11 mutation previously thought to have ataxia with vitamin E deficiency. What the altered direction of therapy in a family with a newly identified a-mannosidase type 1 entails, is left for the reader to imagine. The corresponding reference refers to a paper on stem cell transplant as a definitive treatment option, which will probably not be a treatment option for this family from Islamabad, Pakistan. The paper rather shows that exome sequencing is of use in autosomal recessive disorders and might yield surprises. Continue reading

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.

Figure 1. The candidate gene landscape of the chr2 FAME region. All genes were searched for the number of hits in PubMed for the listed search terms in an automated fashion. As usual in large linkage intervals, only few genes are known in the context of neurological disorders, while most genes are unknown.

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.

By 2020, probably more than 10 million genomes will have been sequenced. Data interpretation rather than data generation will represent the most pressing issue.

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 children of the genomic revolution

Invitation for the Young Investigators workshop in Kiel, August 23rd-25th, 2012

The workshop. We would like to invite all young scientists within the EuroEPINOMICS program for a joint workshop on pediatric epileptology in late August this year. As a spin-off of the EuroEPINOMICS program, we successfully acquired funding for this workshop through the Hamburg Academy of Science. The idea behind this workshop is to bring together young clinicians and researchers working in the field of pediatric epileptology for an intensive exchange of experiences and knowledge. Researchers from different areas will have the chance to meet and learn from each other and to initiate new collaborations and networks.

The sun is setting over the Mornington Peninsula, Australia. Much of the genetic architecture of the epilepsies is still an unknown terra australis that needs young researchers to understand it.

Preliminary Program. We are planning to have talks by young researchers who will present a broad overview and provide insights into recent discoveries on the genetic causes of pediatric epilepsies, the pathophysiological mechanisms and the clinical relevance. Keynote lectures by experienced scientists including Olivier Dulac (“Is pediatric epilepsy research beneficial?”) and Kristien Hens (“Ethical issues in paediatric epilepsy research“) will provide an interesting framework for this meeting. Additionally, all participants may present their current research projects and critically discuss them with their peers. Following this full work program, we will enjoy the summer evenings at the Baltic Sea in Kiel. Here you will find the links to the flyer and the preliminary program for the workshop.

Travel support is granted. We will support participants with the travel expenses and this meeting is without a registration fee thanks to the support of the Academy of Science, University of Kiel and other sponsors. For more information visit our website or contact us on YoungResearchers@epilepsiegenetik.de.

We are looking forward to seeing you in Kiel in August. Don’t miss it!

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.

Study by Lemke et al. demonstrating the usefulness of targeted NGS in patients with epilepsy. Unlike few other genetic technologies, targeted NGS is very likely to alter your work flow in clinic at short term.

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.

Probing autism for hidden autosomal recessive mutations using exome sequencing

Study design applied by Chahrour et al., PLOS Genetics 2012 to identify autosomal recessive genes in non-syndromic autism.

Autosomal recessive neurological disorders are usually distinct and severe diseases that result from the combination of two recessive alleles transmitted by parents. Autosomal recessive disorders are rare, but collectively account for a significant fraction of the genetic morbidity.  With respect to neurodevelopmental disorders including epilepsy, neurometabolic disorders and storage disorders frequently result in complex phenotypes that also comprise intellectual disability, behavioural issues and seizures. Particularly in populations with a high degree of consanguinity such as certain Arab populations, recessive disorders represent a major challenge.

Is autism a recessive disorder? Some recessive disorders might present with atypical phenotypes and are “hypomorphic“. Given that recessive disorders may appear sporadic, i.e. only a single child is affected, it is virtually impossible to distinguish the inheritance pattern in a single individual, particularly in small families. Accordingly, the question frequently arises, if and to what extent neurodevelopmental disorders may either be atypical presentations of known recessive disorders or may be due to novel, as yet unknown recessive mutations. Continue reading