More than half of all epilepsies have some genetic basis. Over the past decade, remarkable advances have been made in our understanding of the genetic causes of many of these epilepsies. To date, most of these advances have been translated into diagnostic tests principally for the rare, severe early-onset epilepsies. We have also learned about several recurrent copy number variations in the genome that increase risk for generalized and focal epilepsies as well as for related neurodevelopmental or neuropsychiatric disorders. As a consequence of these advances, genetic testing is increasingly considered a routine approach in the workup of children with epilepsy. All clinicians who treat children with epilepsy need to be familiar with the principles of genetic testing and counseling.
Genetic evaluations in epilepsy include a review of a patient’s history and medical records, and documentation of at least a three-generation pedigree. Implementation of epilepsy protocol-based MRI of the brain and a sleep EEG are also considered essential for such evaluations. Genetic tests can encompass examination of metabolites and/or enzymatic activities, as well as molecular genetic testing and/or cytogenetic assays (including chromosome microarray analysis). Such tests can require samples from blood, urine, cerebrospinal fluid, muscle or skin. Updated lists of available genetic tests and the laboratories performing these assays are available online. Certified clinical laboratories should be used for patient diagnosis. The results of the clinical investigation, which include the results from genetic testing, are subsequently used in genetic counseling of the patient and their family to identify other family members at risk and to provide informed reproductive decisions (Box 1).
Copy number variants (CNVs) are deleted or duplicated sequences of chromosomal DNA that can be found throughout the genome. Following a large-scale screening of epilepsy patients, the first major CNV discovery in epilepsy was the recurrent deletion of 15q13.3 in individuals with genetic generalised epilepsy (GGE). Approximately 1% of patients with GGE have the 1.5-Mb deletion that includes six genes. The 15q13.3 deletion is also present in individuals with intellectual disability, autism spectrum disorder, and schizophrenia; however, the greatest proportion of affected individuals is found in epilepsy cohorts. Likewise, deletions of 15q11.2 and 16p13.11 are each found in ~1% of patients with GGE, as well as occasionally in individuals with other types of epilepsy.
Whole-genome screening for CNVs has shown that ~5% of individuals have a probable pathogenic deletion or duplication. This is typical for focal epilepsy, generalised epilepsies, epileptic encephalopathies, febrile epilepsy syndromes, and in patients with broadly defined developmental disorders and epilepsy.
The most substantial technological improvement in human genetics research in recent years is the development of next-generation sequencing (NGS). Also known as “massively parallel sequencing,” this process has taken the place of conventional Sanger sequencing in both the research laboratory and in clinical diagnostics. In comparison to Sanger sequencing, where sequencing is done one fragment at a time, NGS can simultaneously sequence millions of short fragments of DNA at once. The entirety of a human genome can be sequenced more quickly and efficiently using this method. Two of the most prevalent applications of NGS are gene panels, which can range from a few to several hundreds of genes, and whole genome sequencing, where the exon sequence of nearly all ~20,000 human genes are sequenced. The coverage for each gene is often preferred in gene panels, although a small percentage of coding sequence is missed in each test.
Exome sequencing is an unbiased method to gene discovery in probands and in families because nearly all protein-coding areas of the genome are examined. An advantage of exome sequencing is the ability to identify the causative mutation in previously unsolved epileptic encephalopathies caused by de novo mutation. Some recently identified genes for EE syndromes include: KCNT1, SCN8A, TBC1D24, GABRA1, GABRB3, ALG13, and DNM1.
Gene panels are pre-selected sets of genes that can be simultaneously sequenced in an individual or a cohort. As fewer genes are sequenced, genes panels cost less than entire exome sequencing and have better coverage of those genes. Originally applied in a research environment, gene panels have been used for gene discovery, including: GRIN2A, GRIN2B, SLC6A1, CHD2, and SYNGAP1. Gene panels that include the most frequently mutated genes can be applied for diagnosis, with a 25-50% diagnostic rate, depending on the panel and patient population.
NGS in Clinical Diagnosis
Gene panels are now widely used in clinical settings and are particularly useful for disorders with substantial genetic heterogeneity. As the amount of identified epilepsy genes continues to expand, and the phenotypes connected to mutations in each gene are variable, gene panel testing is becoming increasingly favourable for diagnosing epilepsy patients. The number of genes included in standard clinical epilepsy gene panels varies significantly, from less than 20 to nearly 500.
Prior to ordering a test, it is important for the clinician to understand which genes or class of genes each panel includes and what the target population is. Most panels are devised for early onset epilepsy syndromes, and a majority strive to be comprehensive; however, if a particular gene or condition is suspected, the clinician should confirm that the selected panel includes the presumed gene or diagnosis. Gene panels for specific subtypes are often accessible, including progressive myoclonic epilepsy and familial focal epilepsy. In some panels, genes for recessive metabolic disorders and congenital disorders of glycosylation are included, whereas others emphasize de novo dominant and X-linked causes.
Depending on the particular phenotype, “nonepilepsy” genes panels for microcephaly syndromes, intellectual disability syndromes, or episodic ataxia may be more applicable. Selecting a company and specific gene panel can be overwhelming, given the variety of choices available. Resources to identify tests include Gene Tests (http://www.genetests.org) and Genetic Testing Registry (https://www.ncbi.nlm.nih.gov/gtr/). It may be useful to consult with a genetic counsellor or epilepsy geneticist before ordering to ensure an efficient and economical testing method.
In gene panels and even single gene tests, the availability of family DNA may be essential for analysing results. For example, it is challenging to define the clinical significance of a rare missense change in a gene for epileptic encephalopathy that has not been previously described in cases or controls. Therefore, testing parents assists in determining whether the mutation is inherited (and therefore likely benign), or de novo and likely causative. In cases of familial epilepsy, the ability to conclude whether a mutation segregates with disease is equally important.
Clinical Utility of Genetic Testing
Often, genetic testing for epilepsies is complex because mutations in a variety of different genes can cause seizures. However, genetic testing can be beneficial in clinical situations by clarifying the prognosis, assisting in treatment choices, and predicting the risk of disease in other family members. “Diagnostic” genetic testing refers to a patient who is symptomatic, whereas “predictive” is a term used for a patient who is asymptomatic, but at risk for developing the disorder in the future.
- Diagnostic testing in a patient with epilepsy
- Confirm a clinical diagnosis of a particular genetic syndrome or epilepsy type
- Differentiate between syndromic and non-syndromic types of epilepsy
- Determine the genetic cause of idiopathic epilepsy
- Provide information about patient prognosis
- Support the selection of optimal treatment options
- Predictive testing for asymptomatic family members of a patient with a known disease-causing mutation that correlates with a genetic form of epilepsy
- Facilitate with clinical monitoring, follow-up, and optimal treatment when symptoms arise in an individual with a positive result
- Reduce apprehension and forego clinical monitoring if result is negative
- Prenatal diagnosis in at-risk pregnancies for established, pathogenic mutations
- Genetic counselling, recurrence risk conclusion, and family planning
Utility of Genetic Testing in Treatment
The treatment of epilepsy is dependent upon the type of seizure, age of patient, and a number of other factors. An understanding of the genetic aetiology of epilepsy may advise selection of the most suitable treatment options in some cases. There are a large number of antiepileptic medications used to treat epilepsy. The selection of an antiepileptic medication for each patient may be determined by the particular type and aetiology of seizures. For example, some medications may be more effective for infantile spasms eg Vigabatrin in tuberous sclerosis. Similarly, certain stiripentol may be indicated for Dravet syndrome, or the ketogenic diet in Glut1 Deficiency. Carbamazepine, oxcarbazepine, phenytoin, lamotrigine and vigabatrin can all induce or increase the frequency of myoclonic seizures in patients with SCN1A-positive Dravet syndrome, and should be avoided.
What Test Should I Send and When?
There are a number of options and resources available for genetic testing in patients with epilepsy. Therefore, it is important to follow some simple guidelines that can help direct the testing strategy.
- There are still indications for testing a single gene, especially in disorders where a single gene is linked to a majority of cases. For example, a single gene test of SCN1A or MECP2 will confirm the diagnosis in patients with Dravet syndrome or Rett syndrome, respectively. However, if a single gene test is negative, the next course of action might be gene panel or aCGH, depending on the phenotype.
- If the patient’s major feature is developmental delay, intellectual disability, autism spectrum disorder, congenital anomalies, etc. and seizures are a part of the phenotype, then aCGH testing should be considered first to detect CNVs. However, in patients with a less severe, drug responsive forms of epilepsy, aCGH testing has not been proven to result in a high diagnostic yield. While never employed for diagnosis, standard karyotypes may be used to confirm aCGH findings. It is worth noting that when Ring 20 syndrome is suspected, karyotype is likely the only testing method available to detect the abnormality.
Whereas the chance of recurrence for a de novo mutation is statistically very low, recessive conditions have a 1:4 risk of recurrence with each pregnancy. Furthermore, dominant conditions have a 50% recurrence risk.
Genetic Test Results and What They Mean
Following genetic testing, three kinds of test results are possible:
Positive: A positive test result means that a disease-causing mutation was found. This result can be used to confirm the diagnosis of a certain epilepsy type and provide the clinician and family with information regarding treatment, prognosis, and risk of recurrence. Once a patient has a positive test result, predictive testing can be offered for all first-degree relatives. In the case that a family member is positive for the familial mutation, they are at increased risk for developing epilepsy and should be referred for further monitoring. Moreover, among family members with the same genetic mutation, there is often variability in age of onset, symptoms, severity, and response to therapeutic agents.
Negative: A negative test result can have several explanations. These may be:
- the patient may have a mutation in a gene that is not part of the testing panel
- the patient may have a mutation in a part of an epilepsy gene that was not tested
- the patient does not have a heritable type of epilepsy
In an individual with epilepsy, a negative result from a specific genetic test means that predictive asymptomatic family members will not require that same test. However, family members of an epilepsy patient may still be at risk for developing epilepsy syndromes and should be examined by a neurologist if necessary.
A result is considered a “true negative” in the case that an asymptomatic individual is negative for a mutation found in a family member with epilepsy. This means that the individual is not at increased genetic risk for the familial epilepsy syndrome, rather, they have the same chance of developing epilepsy as any individual in the general population. It is not necessary to clinically monitor the development of seizures in a person with a “true negative” genetic result.
Variant of Unknown Significance: The most challenging result to interpret is VUS because it indicates that the pathogenic role of the variant cannot be definitively established. Testing other family members may be useful in interpreting the clinical significance of a VUS. If other relatives with epilepsy have the same variant, it is probable that the variant is disease-causing. There is a greater chance that the VUS is pathogenic if more affected family members carry the VUS. Similarly, if a patient has sporadic epilepsy, the finding of a de novo VUS means that the variant is likely disease-causing.
Genetic testing has an established clinical utility in the diagnosis and management of certain epilepsies, and will increase in importance in other epilepsy syndromes over the next few years. For example, in conditions such as Dravet syndrome, which is no longer considered rare, tests can guide specific treatment indications and contraindications, and the pharmacological management of status epilepticus. In early onset epileptic encephalopathies, gene panel or exome sequencing offer high diagnostic yield. aCGH also has a role in the diagnosis of selected epilepsies and for neurodevelopmental conditions that include a seizure phenotype.