Pathogenic EFHC1 mutations are tolerated in healthy individuals dependent on reported ancestry

It has been reported that coding mutations in the EFHC1 (for EF-hand containing) gene are the cause of perhaps up to 9% of all cases of juvenile myoclonic epilepsy (JME).1 The major evidence supporting this conclusion includes the following: (1) Hispanic and Dutch families with epilepsy show linkage to a locus that includes EFHC1,2–6 (2) highly penetrant EFHC1 coding mutations segregate with JME in families reporting Hispanic and Central American ancestry,7 (3) rare EFHC1 mutations have been reported to be both inherited and de novo in sporadic JME cases,8 (4) Efhc1-deficent mice exhibit spontaneous seizures,9 (5) EFHC1 regulates important neurodevelopmental events in cell culture.10,11 However, despite this evidence, notable contradictions still remain. For instance, in the study that initially proposed mutations in EFHC1 as a cause of JME,7 four separate coding mutations were found to segregate in 6 of the 30 JME families that showed linkage to the EFHC1-containing 6p12-p11 locus. Although these four mutations were not found in 382 Hispanic controls, neither they nor any other EFCH1 coding mutations were found segregating in the other 24 families in the study. Therefore, the six families were the only families in which these four EFHC1 mutations were found to segregate with JME. It is notable that when these six families were excluded from the analysis, evidence in favor of linkage remained.7 Although these observations support the hypothesis of a JME gene existing in the region, they cast some doubt on the importance of EFHC1 coding mutations for disease and/or on the role of EFHC1 in JME susceptibility. A subsequent study of Dutch JME patients also found linkage to the same region5 but failed to find any exonic mutations in EFHC1 associated with JME.12 Thus, in the majority of 6p12-p11-linked JME families from both Hispanic and Dutch populations, EFHC1 coding mutations do not segregate with disease. This means that other elements in the region, either within EFHC1 or in some other gene, must be responsible for the genetic signal. These elements might include regulatory factors affecting EFHC1. However, an additional possibility that cannot be ruled out is that EFHC1 is not the actual causative gene and that mutations in some unexamined nearby element functionally unrelated to EHFC1 are the cause of 6p12-p11-linked JME. One example that illustrates how causal mutations can be in a gene nearby a gene initially thought to be causal is in benign familial neonatal seizures (BFNS). Initially, given the observation of a nonsense mutation in CHRNA4, the gene encoding the neuronal nicotinic cholinergic receptor α4 subunit, segregating13 with BFNS in a linked region of chromosome 20q,14 this mutation was thought to be causal. At the time of its discovery, the evidence seemed convincing. However, it was ultimately shown that mutations in a nearby gene KCNQ2 were in fact the cause of 20q-linked BFNS.15,16 Thus, whether one considers CHRNA4 and BFNS or EFHC1 and JME, the existence of segregating mutations is not proof of causality, especially in the case of EFHC1, which also happens to have several coding mutations that are unrelated to epilepsy.7,17,18 (A fact often ignored in a cosegregation argument is that, in a linked region, all variants within the region, including those not in the disease gene itself, are more likely to cosegregate with the disease, by the definition of linkage analysis. This means that cosegregation alone cannot prove that a variant is the cause of the disease.) Importantly, if it is the case that another gene at 6p11-12 is causal for JME, the mutations are unlikely to be coding mutations, as mutation analysis of several genes in the region seems to rule out this possibility.18 More recently, exome-wide sequencing efforts have raised another issue regarding the role of EFHC1 in JME. Specifically, two of the four mutations initially reported to cause JME with high penetrance, and reported to co-segregate with JME,7 are variants found in individuals in the National Heart, Blood and Lung Institute Exome Variant Server (EVS: http://evs.gs.washington.edu/EVS) database.19 The EFHC1 F229L variant (rs137852776) is relatively rare (0.4%) and carriers in the EVS database could possibly be JME patients, since the sample is unphenotyped. However, the frequency of the other variant, the EFHC1 P77T-R221H haplotype (rs149055334-rs79761183), is found in >2% in the EVS data base among African Americans, a surprisingly high frequency for a haplotype reported to cause JME with roughly 80% penetrance. Without phenotypic data on these individuals, we cannot rule out the possibility that the EVS individuals with this haplotype are also JME patients, although a frequency of >2% significantly exceeds the prevalence of all forms of JME in any population. Therefore, an important question becomes, for example, “What does it mean to be an African American carrying the EFHC1 P77T-R221H allele and how does this impact individual susceptibility to epilepsy?” The reported role of pathogenic EFHC1 variants in JME7,8 has been mentioned in reports weighing the benefits and harm of genetic testing in the epilepsies (e.g., Ottman et al.20), with the causative role of EFHC1 in JME listed as one of the most well-accepted findings. Ottman et al. note that being told that one carries a risk allele for epilepsy can lead to anxiety, stigmatization, and discrimination. Therefore, it is important to distinguish between “susceptibility” alleles and “causal” alleles. “Susceptibility” alleles interact with other genetic or environmental factors to increase the chances of developing disease, but they need not have any direct effect on the underlying disease mechanism and are not necessary for the disease to manifest. In contrast, the defining characteristic of “causal” alleles is their clear direct involvement in the disease mechanism with interactions between other genetic or environmental factors being of secondary importance in disease expression. If the putative EFHC1 disease alleles are in fact causative, as is the case for genes involved in several severe, monogenic forms of epilepsy (e.g., SCN1A in Dravet syndrome21), their interpretation in the clinic will be substantially different than if they are merely susceptibility alleles. Therefore, it is essential that the risk attributed to genetic variants not be overstated. Furthermore, understanding which category EFHC1 fits into is of importance in understanding the mechanisms underlying epilepsy susceptibility. This is especially important because, unlike the ion channel–encoding candidate genes, which have been studied extensively in epileptogenesis, EFHC1 appears to function in neural development and differentiation,22,23 a mechanism for epileptogenesis only recently being widely acknowledged.1,24 Because of the need to properly assess the effect of EFHC1 disease alleles in JME, we investigated the reportedly strong effect of EFHC1 coding mutations in Hispanic patients with epilepsy, where the effect is presumed to be particularly large. Our controls were carefully selected so as to have no family history of epilepsy (as opposed to the unphenotyped individuals in the EVS [http://evs.gs.washington.edu/EVS] and 1,000 Genomes databases [http://www.1000genomes.org]. We measured the frequency of the reportedly pathogenic EFHC1 P77T-R221H and F229L alleles in specific populations and used 1,000 Genomes data to supplement our findings. Our study does not find an excess of EFHC1 mutations in JME patients, but may suggest a possible ancestry-specific component governing susceptibility to EFHC1-based JME. Such possible interactions must be considered when assigning risk values to susceptibility alleles in JME and in all complex disorders, especially when considering such variants in applications such as designing genetic testing panels for assessing risk for a particular disease.