SCN1A Seizure Disorders are generally considered to have a broad phenotype ranging from simple febrile seizures and generalized epilepsy with febrile seizures plus to Dravet syndrome and retractable childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC)24. However, the distribution ratio of these phenotypes in SCN1A Seizure Disorders is unclear due to the limited number of cases in various institutions. So far, ClinVar has included 1360 variations related to SCN1A Seizure Disorders. When CNV is removed and only single-nucleotide variations are retained, there are 1,139 variations being screened. Among these ClinVar variants, 1* variants provided by a single submitter have lower associated evidence than 2* or 3* variants25. At the same time, not all of the conditions of SCN1A Seizure Disorders shown by ClinVar have case reports. The evidences for some conditions are insufficient to support the correlation of the target variants with the conditions. Therefore, in our study, ClinVar P/LP SCN1A missense variants provided by a single submitter were reclassified, and then these reclassified conditions of SCN1A Seizure Disorders were validated by querying case reports in Pubmed.
1. Reclassification of ClinVar P/LP SCN1A missense variants provided by a singgle submitter
Reclassification outcomes of 383 ClinVar P/LP SCN1A missense variants provided by a single submitter shows that most of the P/LP variants remained at the P/LP level (247/383)(Table.1), and a few variants were converted from P/LP to VUS (136/383)(Table.1). Among them, there are 10 variants changed from P/LP to VUS due to the reduced level of PS2 evidence (Supplementary Table 1), including p.Met1348Ile, p.Pro657Leu, p.Ser626Gly, p.Val422Ala, p.Met350Val, p.Ala342Ser, p.Ala333Thr, p.Val250Ile, p.Asn115Lys and p.Ala24Thr. According to specifications for the ACMG/AMP recommendations developed by the ClinGen Sequence Variant Interpretation Working Group (SVI) and various disease-specific variant curation expert panels (VCEP) (https://clinicalgenome.org/working-groups/sequence-variant-interpretation/), when the phenotype is consistent with the gene but not highly specific and if there is a de novo status with validated parental relationships, the PS2 evidence strength level is 1 point (PS2_Moderate); if there is a de novo status with unvalidated parental relationships, the PS2 evidence strength level is 0.5 points (PS2_Supporting). Seizure is not a highly specific phenotype26.Therefore,PS2_Moderate is applied according to the SVI recommendation for de novo criteria.
In addition, p. Gly682Val (Supplementary Table 1) changed from the PS3 level to PS3_Supporting, resulting in a change of P/LP to VUS. According to recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework27, if the number of benign/pathogenic variant controls used in a functional study is less than 10, PS3 should be changed to PS3_Supporting. Regarding p.Gly682Val, as only one pathogenic variant was used and a benign variant was not used as controls, PS3_Supporting fits the pathogenicity assessment for this variant according to the SVI recommendation.
Besides, the assignment of p. Asp1742Asn (Supplementary Table 1) is changed from P/LP to VUS due to reduced PM2 levels. Based on SVI Recommendation for Absence/Rarity (PM2) (https://www.clinicalgenome.org/site/assets/files/5182/pm2_-_svi_recommendation_-_approved_sept2020.pdf), the weight of criterion PM2 has been changed from a moderate level to a supporting level (PM2_Supporting), which resulted in a change in the classification of p.Asp1742Asn.
2. Validation of conditions of SCN1A Seizure Disorders in ClinVar and statistical analysis of variant types of conditions of SCN1A Seisure Disorders by validation
Validation outcomes of variants related to the conditions of SCN1A Seizure Disorders in ClinVar suggests that the condition "Early infantile epileptic encephalopathy with suppression bursts" shown in ClinVar is actually all other conditons, such as Dravet syndrome/Severe myoclonic epilepsy of infancy( 69/89) and Generalized epilepsy with febrile seizures plus/Borderline severe myoclonic epilepsy of infancy (3/89)(Supplementary Table 2). Among all conditions of SCN1A Seizure Disorders, DS/SMEI accounts for the highest proportion (291/371)(Table.2), which is much higher than other conditions. GEFS+/SMEB accounts for a lower proportion (19/371)(Table.2). In addition, There are 35 SCN1A gene variants having Phenotype heterogeneity of varying severity and there are 26 SCN1A gene variants associated with rare conditions such as familial hemiplegic migraine(Table.2).
According to the Gene-Disease Validity presented by CinGen(URL: https://clinicalgenome.org/), the SCN1A gene is classified as "Definitive" in generalized epilepsy with febrile seizures plus, "Definitive" in Dravet syndrome, "Strong" in developmental and epileptic encephalopathy and "Moderate" in familial hemiplegic migraine. The OMIM(URL: https://www.omim.org/) database suggests that the SCN1A gene is associated with Developmental and epileptic encephalopathy 6B, non-Dravet, Dravet syndrome, February seizures, familial, 3A, Generalized epilepsy with febrile seizures plus, type 2 and Migraine, familial hemiplegic, 3. No relevant database is found showing the association of SCN1A gene with Early infantile epileptic encephalopathy with suppression bursts. Therefore, the condition "Early infantile epileptic encephalopathy with suppression bursts" submitted in ClinVar is suspected to be excluded from SCN1A Seizure Disorders.
Distribution of variant types of the conditions of SCN1A Seizure Disorder in ClinVar by validation shows that among all truncation variants, DS/SMEI-related SCN1A variants account for the vast majority (151/170, 88.8%)(Figure.2B). This is consistent with the fact that the pathogenic mechanism of DS/SMEI-related SCN1A variants is loss of function28,29.Severe Myoclonic Epilepsy in infancy (SMEI, or Dravet syndrome) is a severe condition on the genetic epilepsy with febrile seizures plus (GEFS+) spectrum of seizure disorders30,31. The clinical presentation of DS/SMEI is characterized by frequent febrile seizures followed by non-febrile seizures, mainly clonic and unilateral, of prolonged duration and frequent status epilepticus32. There is sufficient case-level and experimental data evidence to support this gene-disease pair29,33−36. In addition, expression, protein interaction, and biochemical function studies as well as mouse models also provide experimental evidence that SCN1A variants cause DS37,38.
Generalized epilepsy with febrile seizures plus (GEFS+) is a rare autosomal dominant, familial condition with incomplete penetrance and variable expressivity, which includes seizures ranging from usually mild (eg, isolated febrile seizures) to less severe (including medically treatable generalized epilepsy, refractory generalized epilepsy, or in some cases Dravet syndrome)39. The penetrance of the GEFS + phenotype has been estimated to be 70%40.In affected families, parents of more than 95% of individuals with GEFS + also have the same SCN1A pathogenic variant41. Distribution of variant types of the conditions of SCN1A Seizure Disorder in ClinVar by validation shows that the number of variants in GEFS+/SMEB is low (5.2%)(Table.2), indicating that although SCN1A variants have conditions ranging from mild to severe, variants associated with severe conditions are still the mainstream of SCN1A variants.
Analysis of variants related to rare conditions of SCN1A Seizure Disorder in ClinVar by validation shows that p.Val422Leu(c.1264G > T ), p.Pro1345Ser(c.4033C > T ) and p.Thr226Met (c.677C > T) are all associated with developmental and epileptic encephalopathy (DEE)(Supplementary Table 5). Distinguished by DS/SMEI, DEE has a younger age at onset, beginning at < 3 months compared with the typical seizure onset age range of 4 to 15 months in Dravet syndrome19. Unlike DS/SMEI, DEE has Severe developmental disability and reported movement disorders including choreoathetosis, dystonia, and perioral hyperkinesia. Currently the 3 SCN1A variants that have been reported to be associated with DEE42 are all missense variants, the most notable of which is p.Thr226Met (c.677C > T)(Table.4), a variant that has been identified in several individuals with similar characteristics and identified as de novo19,43,44. Because these three SCN1A variants is discovered for a short time, the association of SCN1A with DEE is not well documented. It is currently known that the mechanism by which SCN1A variation leads to the phenotype of DEE is mainly due to gain of function42.
Analysis of variants related to rare conditions of SCN1A Seizure Disorder in ClinVar by validation shows that three missense variants in SCN1A have been reported to associated with familial hemiplegic migraine (FHM) ( Supplementary Table 5,Table.4), including one de novo variant45,46. FHM is a rare autosomal dominant disorder characterized by migraine attacks with aura and associated hemiplegic attacks, in which SCN1A-associated FHM has a penetrance approaching 100%47.The recognized mechanism of aura in hemiplegic migraine is cortical spreading depression. Because the effects of FHM variants in SCN1A are often complex and diverse, the exact mechanism by which defects in SCN1A may lead to cortical spreading inhibition is unknown. According to ClinGen's gene curation classification, the association between SCN1A and FHM is "Moderate", which means that the pathogenic classification of all SCN1A variants associated with FHM can only be classified as "Likely pathogenic" at the highest level.
Both in missense and trunction, DS/SMEI-related SCN1A variants have the highest proportion (291/371, 78.4%)(Figure.2B), which shows that although DS/SMEI is the most important condition in SCN1A Seizure Disorders, there are still quite a few some SCN1A variants are associated with other conditions such as GEFS+, FMH, DEE, Lennox-Gastaut Syndrome and Intractable childhood epilepsy with generlaized tonic-clonic seizures (80/371, 21.6%)(Supplementary Table 5).