Genetic Risk Factors for Epilepsy: From Familial Studies to Gene Discoveries and Polygenic Risk Scores, Strides Toward Unlocking an Age-Old Question in Epilepsy

Abstract

Article 1: Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here, we report a screen for de novo mutations in patients with 2 classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox-Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ∼4000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 × 10(−3)). Among these are GABRB3, with de novo mutations in 4 patients, and ALG13, with the same de novo mutation in 2 patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10(−10) and P = 7.8 × 10(−12), respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR, and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P < 10(−8)), as has been reported previously for autism spectrum disorders.

Article 2: Identifying genetic risk factors for highly heterogeneous disorders such as epilepsy remains challenging. Here, we present, to our knowledge, the largest whole-exome sequencing study of epilepsy to date, with more than 54,000 human exomes, comprising 20,979 deeply phenotyped patients from multiple genetic ancestry groups with diverse epilepsy subtypes and 33,444 controls, to investigate rare variants that confer disease risk. These analyses implicate 7 individual genes, 3 gene sets and 4 copy number variants at exome-wide significance. Genes encoding ion channels show strong association with multiple epilepsy subtypes, including epileptic encephalopathies and generalized and focal epilepsies, whereas most other gene discoveries are subtype specific, highlighting distinct genetic contributions to different epilepsies. Combining results from rare single-nucleotide/short insertion and deletion variants, copy number variants and common variants, we offer an expanded view of the genetic architecture of epilepsy, with growing evidence of convergence among different genetic risk loci on the same genes. Top candidate genes are enriched for roles in synaptic transmission and neuronal excitability, particularly postnatally and in the neocortex. We also identify shared rare variant risk between epilepsy and other neurodevelopmental disorders. Our data can be accessed via an interactive browser, hopefully facilitating diagnostic efforts and accelerating the development of follow-up studies.

Article 3: Background: The epilepsies are highly heritable conditions that commonly follow complex inheritance. While monogenic causes have been identified in rare familial epilepsies, most familial epilepsies remain unsolved. We aimed to determine (1) whether common genetic variation contributes to familial epilepsy risk, and (2) whether that genetic risk is enriched in familial compared with nonfamilial (sporadic) epilepsies. Methods: Using common variants derived from the largest epilepsy genome-wide association study, we calculated polygenic risk scores (PRS) for patients with familial epilepsy (n = 1818 from 1181 families), their unaffected relatives (n = 771), sporadic patients (n = 1182), and population controls (n = 15,929). We also calculated separate PRS for genetic generalized epilepsy (GGE) and focal epilepsy. Statistical analyses used mixed-effects regression models to account for familial relatedness, sex, and ancestry. Findings: Patients with familial epilepsies had higher epilepsy PRS compared to population controls (OR 1·20, p_adj = 5 × 10⁻⁹), sporadic patients (OR 1·11, p_adj = 0.008), and their own unaffected relatives (OR 1·12, p_adj = 0.01). The top 1% of the PRS distribution was enriched 3.8-fold for individuals with familial epilepsy when compared to the lowest decile (p_adj = 5 × 10⁻¹¹). Familial PRS enrichment was consistent across epilepsy type; overall, polygenic risk was greatest for the GGE clinical group. There was no significant PRS difference in familial cases with established rare variant genetic etiologies compared to unsolved familial cases. Interpretation: The aggregate effects of common genetic variants, measured as PRS, play an important role in explaining why some families develop epilepsy, why specific family members are affected while their relatives are not, and why families manifest specific epilepsy types. Polygenic risk contributes to the complex inheritance of the epilepsies, including in individuals with a known genetic etiology.

Commentary

Is epilepsy a genetic disorder? If so, who should be tested? An age-old question that has been asked by and challenged many generations of neurologists. In past decades, the answers might have been “probably” and “only infants with early-onset epilepsy” or “patients with a strong family history of epilepsy.” Advances in the scientific understanding of genetic causes of epilepsy have since revolutionized clinical diagnosis. In 2022, the American Epilepsy Society endorsed an evidence-based practice guideline from the National Society of Genetic Counselors that recommends genetic testing for “all individuals with unexplained epilepsy, without limitation of age.” ¹ The National Institute of Neurological Disorders and Stroke (NINDS)-funded Epi4K and Epilepsy Phenome/Genome studies were instrumental in reaching this new clinical understanding. In this commentary, we highlight 3 of the many papers presenting genetic discoveries from these projects.

Epilepsy has been suspected and postulated to have a genetic cause since before the molecular genomic era. Investigations from the twin study by William Lennox to cohort studies of families with multigenerational epilepsies shed light into the genetic risk factors of epilepsy. But not until 1995, when the first “epilepsy gene,” CHRNA4, was identified in a large family with frontal lobe epilepsy was the concept that variants in a single gene (monogenic) can cause epilepsy solidified. Since then, there has been rapid progress of epilepsy gene discoveries, at least for the monogenic epilepsies, thanks to advances in molecular genomic technology such as next-generation sequencing leading to exome/genome testing. However, the genetic causes of common epilepsies, such as genetic generalized epilepsy (GGE), remain difficult to find despite indisputable evidence of familial risk.

Beginning with monogenic epilepsies, we now know that most presentations of developmental-epileptic encephalopathy (DEE) are phenotypes of monogenic disorders affecting any of several hundred genes. In “De novo mutations in epileptic encephalopathies,” ² Epi4K and Epilepsy Phenome/Genome laid the groundwork for this new understanding by searching the exomes of patients with infantile spasms (n = 149) and Lennox-Gastaut syndrome (n = 115) for de novo variants not present in their parents. Although not all de novo variants are disease-causing, this enriched for likelihood to cause disease. Variants were then grouped for analysis based on occurrence in the same gene or defined sets, such as evolutionarily conserved genes and ion channels, making it possible to do statistical analyses of ultra-rare variants that otherwise would have had sample sizes of one or zero. Epi4K found that the distribution of de novo variants in DEE was significantly shifted toward loss-of-function variants, evolutionarily conserved genes intolerant to variation, and genes in 6 selected neurologically relevant sets. Analysis of single genes confirmed significantly higher de novo variation in then-known DEE genes such as SCN1A and STXBP1 and discovered new DEE-related genes such as GABRB3. These findings indicated that DEEs really represented shared phenotypes of many, individually rare, monogenic disorders. In the clinic, this means physicians sending genetic testing should cast a wide net among hundreds of DEE-causing genes, with numerous studies supporting exome or genome sequencing as the most cost-effective initial test. ³

This paradigm shift extends outside DEEs: all nonlesional epilepsies have since been found to have contributions from ultra-rare genetic variants that improve treatment by allowing gene-tailored treatment and prognostication. In the subsequent, larger Epi25 study, grouping variants by gene and gene set revealed significant associations with not only DEE but also GGE and nonacquired focal epilepsy (NAFE) in “Exome sequencing of 20,979 individuals with epilepsy reveals shared and distinct ultra-rare genetic risk across disorder subtypes.” ⁴ Interestingly, variants affecting ion channels like GABA_A receptors were associated with epilepsies across subtypes, while others were putatively associated with specific subtypes, that is, phosphodiesterase gene family with NAFE. Among adults with unexplained epilepsy, real-world application of exome sequencing with mitochondrial DNA analysis at the tertiary epilepsy center of the Medical University of Vienna revealed rare monogenic epilepsy diagnoses in 30.2%, a quarter of which had clinical implications including tailored treatment for SCN1A and SLC2A1-related disorders and other organ-system screening in mitochondrial disease. ⁵

What about oligogenic or polygenic epilepsies with unknown etiologies? Even among known monogenic epilepsies such as SCN1A-related Dravet syndrome/GEFS+, the epileptologist often encounters variable severity in relatives with the same variant, suggesting other contributing genetic/environmental factors. Moreover, some known “causative” results on epilepsy genetic testing have small effect sizes better categorized as risk factors. The recurrent 15q11.2 BP1-BP2 microdeletion, originally described in a neurodevelopmental-epilepsy syndrome, was subsequently found to have an odds ratio (OR) for epilepsy of 3.1, translating to a prevalence of 2.1% in the study population—therefore, most people with this variant did not have epilepsy. ⁶ In “Common risk variants for epilepsy are enriched in families previously targeted for rare monogenic variant discovery,” ⁷ an Epi4K collaboration examined the contribution of common variants with small individual effects by assembling a polygenic risk score (PRS) for epilepsy, which summed the contributions of variants previously discovered as affecting epilepsy risk in genome-wide association studies (GWAS). Surprisingly, PRS was significantly associated with familial and sporadic epilepsy not only across epilepsy types but even in patients with known monogenic epilepsies. In other words, adding together the impact of common, low-effect size variants helps explain epilepsy itself as well as why we see greater expression of seizures in some families than other. For example, in GGE, the PRS displayed significant association with both familial (OR 1.58) and sporadic (OR 1.46) cases compared to the general population. Practicing neurologists do not need to start calculating our patients’ PRS just yet, however, because the effect size is still too weak to reliably predict epilepsy. Although people with familial epilepsy had significantly higher PRS on average, having the highest-centile PRS scores only increased the odds of familial epilepsy by 4-fold, reminiscent of the 15q11.2 microdeletion above. This is primarily because of the limited power of existing GWAS used to create the PRS, The more genetic variants associated with small changes in epilepsy risk that are discovered, the more useful the PRS will become in predicting epilepsy.

Where will the next advances in epilepsy genetics come from? Collaborative efforts like Epi25 lead the way in raising sample sizes. Another crucial improvement to the power of genetic association studies will be capturing genetic diversity from global ancestry populations outside the 79% European-ancestry cohort in the Epi25 paper discussed above. The epilepsy research community should take inspiration from genomic projects like the NHGRI-funded All of Us Research Program, which is successfully capturing diversity across Americans with 46% participants from underrepresented minorities.^8,9 Understanding which genetic variants affect gene function could also raise power by filtering out benign variants. Machine-learning algorithms for in silico prediction such as REVEL, in common use among geneticists, are useful in this task; it is, however, important to remember that they never suffice to determine pathogenicity on their own and fancy new “AI” tools such as AlphaMissense have only contributed modest if any improvements so far. ¹⁰ Experimentally measuring the functional impacts of thousands of genetic variants in vitro is becoming feasible both through robotic systems and new genetic techniques such as MAVE (multiplexed assays of variant effect), recently applied to cardiac ion channelopathies. ¹¹ Finally, several lines of research are improving our ability to detect genetic variants. Geneticists consider “whole” exome or genome sequencing an inappropriate term ¹² because data on large chunks of the genome that are repetitive, structurally variable, or otherwise hard to sequence are missing. The scale of this issue is broad: the current human reference genome, GRCh38, actually lacks >6% (200 million bases) of its sequence. ¹³ Technical and conceptual improvements to the reference human genome by the Telomere-to-Telomere and Human Pangenome Reference consortia will allow variants in these “missing” regions to start being detected and studied. Long-read sequencing technologies that better capture repeat expansions are enabling the discovery of new diseases like large intronic pentanucleotide repeat expansions in SAMD12, TNRC6A, and RAPGEF2 in adult familial myoclonic epilepsy. ¹⁴ Finally, work on somatic variants is starting to provide genetic explanations for even lesional epilepsies. Using deep sequencing of brain tissue, one such NINDS-funded study discovered that somatic SLC35A2 variants can cause both focal cortical dysplasias and nonlesional focal epilepsy. ¹⁵

We now know genetics plays pivotal roles in epilepsy, as a cause, a risk factor, and a strong contribution to phenotypic variability. However, many questions about the genetics of epilepsy remain unanswered. With further advances in molecular genomic technology, a collaborative work and tireless effort from neurologists and scientists, and support from professional and research organizations, we may one day cease to ask the age-old question on whether and how genetics cause epilepsy.