Annotation Guidelines

Definition of Epistemic Gap

An Epistemic Gap (or Goal for Knowledge) statement identifies a missing or incomplete piece of knowledge requiring scientific investigation to resolve. As such, it provides a target for future research aimed at generating new knowledge.

Types of Epistemic Gap Statements

(i) Gap-oriented: acknowledge a research relevant gap in current scientific understanding by highlighting aspects that are unknown, poorly understood, or understudied.

(ii) Goal-oriented: forward-looking statements that identify scientific goals for future investigation with the aim to advance scientific understanding and generate new knowledge.

Examples of Gap-oriented statements:

“Disruptions in the MBD5 gene have been linked with an array of clinical features such as global developmental delay, intellectual disability, autistic-like symptoms, and seizures, through unclear mechanisms.”

”Though this association is known, the exact mechanisms through which perturbed MBD5 expression leads to neurodevelopmental disorders remain highly underexplored.”

“It is not known how closely the transcriptomic changes in this animal model of Dravet syndrome recapitulate the transcriptomic changes in people with Dravet syndrome.”

“Regarding behavioral disorders, data from the literature may be misleading. Few authors have properly analyzed these features, reported as aggressive or oppositional behaviors with psychiatric traits.” - (Highlights a poorly studied aspect)

“Despite frequent use of mTOR inhibitors, there are only a few studies on their effect on epilepsy control in children under 2-years of age.” - (highlights an understudied area)

“Homomeric GIRK2 channels are predominant in some midbrain nuclei (L\u00fcscher and Slesinger, 2010; Luj\u00e1n and Aguado, 2015), but it is not clear how abundant they are in other brain structures: GIRK2 subunit is ubiquitously expressed throughout the brain, but it is usually co-localized with GIRK1 or GIRK3 subunits, and the proportion of GIRK2 homomers vs. GIRK1/2 or GIRK2/3 heterotetramers remains unknown.”

“Various ASMs were trialed in the literature, with different combinations, making it difficult to draw conclusions regarding which ASM is best for ST3GAL3-related DEE.”

Examples of Goal-oriented statements:

“Larger case series are needed, however, to determine which ASMs are most effective for ST3GAL3-related DEE.”

”The mechanism underlying the link between MBD5 and primary cilia requires further understanding.”

“Second, the functional verification on the animal model should be subsequently conducted to ascertain whether the p.Asp920Glu variant influences the PCDH19 protein function and PCDH19-FE phenotype.”

“Therefore, further elucidation of the role of miRNAs in EP may favor EP diagnosis and treatment.”

“Whether this was meaningful should be further explored by functional experiments.”

"Further research is encouraged to confirm the discovered variants and other mutations as possible epilepsy pathogenic in the larger population.”

“The significance of the brainstem changes is unclear and requires further investigation.”

Exception #1

Not all forward-looking statements qualify for annotation!

Statements that provide future directions for action without identifying a scientific research goal should be annotated as practical recommendations, NOT as research directives. The intent is NOT to generate knowledge that advances scientific understanding, but rather apply existing knowledge (e.g., to support clinical practice, or methodological improvement, etc).

Examples of Practical Recommendations:

❌ “For clinicians, patients with developmental regression accompanied with concurrent epilepsy and autism spectrum disorder should be advised that relevant genetic tests are necessary to clarify the diagnosis.”
(Intent: recommends action for clinical practice, not research directive)

❌ "Despite their caveats, when combined, these methods nevertheless provide a strong means for assessing the functionality of KCC2, albeit, in possible future studies on the functionality of the variant, additional complementary methods should be considered."
(Intent: suggest to improve methodology for variant studies, not indicate the target for future research)

❌ "It becomes apparent that future in vitro studies on KCC2 variants should utilize isolated primary neurons or cortical neurons derived from induced pluripotent stem cells (iPSCs) as model systems."
(Intent: suggest better experimental models, not indicate a specific research goal)

❌ "Future studies should include additional control cell lines to ensure that no off-target effects from gene editing are contributing to the phenotypes observed."
(Intent: improve experimental rigor, not identify what we need to investigated)

❌ "Future endeavors should consider models more apt for matched datasets."
(Intent: improve statistical approach, does not identify a scientific goal for knowledge)

❌ According to this article's findings and previous research, further diagnostic testing and management are needed to determine the severity and progression of the patient's condition."
(Intent: knowledge application to specific case management, not a research directive)

❌ Among the remaining patients with neuroimaging abnormalities, FCD was the common presentation of NPRL3-related epilepsy (62.5%), which was consistent with the previous studies (6). However, for those with negative brain MRI results, small FCD may be overlooked. Thus, a PET scan is needed in the future to reveal the subtle metabolic or biochemical function changes in the brain of patients with NPRL3-related epilepsy.
(This is a clinical/diagnostic action, not research directive. The intent is to suggest a practical next: step use PET scans to mprove detection/diagnosis in patients with negative MRI. This is about what clinicians should do, not what scientists should investigate)

❌ Other conditions that implicate disease severity include Status Epilepticus [94], characterized by continuous or recurrent seizures lasting for an extended period or seizures occurring in rapid succession without the person regaining consciousness in between. Status epilepticus requires immediate medical attention as it can lead to significant complications and neurological damage. Epileptic Encephalopathy [95] refers to another group of severe epilepsy disorders that are associated with cognitive and developmental impairments.

Forward-looking statements with a clear scientific research goal should be annotated as Goal for Knowledge.

Examples:

✅ We still need more detailed and accurate case reports, which will help in investigating the correlation between NUS1 variants and disease prognosis [research goal], and will also lay a solid foundation for the development of accurate and rapid genetic diagnosis and treatment in the future.

✅ Other scalable experimental approaches such as computational neuronal action potential modeling (Ben-Shalom et al., 2022) or dynamic action potential clamp recording (Berecki et al., 2018) may be valuable to determine the net effect of mixed dysfunctional properties on neuronal excitability [research goal].

✅ Larger case series are needed, however, to determine which ASMs are most effective for ST3GAL3-related DEE [research goal].

✅ Longitudinal follow-up is needed into adulthood however to determine how the epilepsy evolves [research goal].

Decision Rule:

Ask: Does the forward-looking statement identify a scientific research goal?

Yes → Goal for Knowledge (research directive) ✅

No → Probably a Practical recommendation ❌

Exception #2

Speculation alone is not sufficient to indicate a goal for knowledge!

Rationale: Scientific writing frequently contains tentative or hedged statements (e.g., may, possibly, suggests) due to disciplinary norms favoring cautious claims. However, not all uncertainty reflects a need for scientific investigation. Most speculative statements do not imply a need for further research. Annotating all such statements as epistemic gaps would lead to a high number of false positives, diluting truly meaningful goals for knowledge annotations.

A speculative statement should be annotated as a scientific goal for knowledge ONLY when it is commited to a testable hypothesis and explicitly framed as a target for future research.

Operational Assumption

When authors intend to propose a genuine research hypothesis for further investigation, they frame it as a future research target using explicit “research goal” markers, such as:

highlight inadequate scientific support (inadequate evidence base to support the claim)

calls for further investigation seaking confirmation

Examples:

✅ These observations raise the possibility that D1050V disrupts NaV1.2 interactions with ankyrin and mimics a channel LoF. [Speculation] Future investigations of the cell surface expression or localization of SCN2A variants in neurons can test this hypothesis. [EXPLICIT CALL FOR RESEARCH]

✅ Thus, loss of function of Bassoon was potentially the underlying mechanism of the pathogenicity of BSN variants, which is consistent with the pathogenesis of other genes encoding CAZ proteins, such as UNC13A\n27 and UNC13B.6 [Speculation] However, the functional effects of the BSN variants warrant further investigation. [EXPLICIT CALL FOR RESEARCH]

✅ Altogether, this could suggest that haploinsufficiency of MBD5 could lead to decreased NMDAR expression, which would have similar effects to an antagonist and thus lead to primary cilia shortening. [Speculation] This should be further explored using high-resolution microscopy and advanced realistic models of human brain development, such as brain organoids. [EXPLICIT CALL FOR RESEARCH]

✅ Two possible hypotheses arise from our findings in this study. The first is that a loss of neuronal homeostasis is due to slowed or lack of production of neuronal proteins. Neurons have a large bias toward very long transcripts (27), so it is possible that the slowed elongation rate caused by the EEF1A2 mutations results in changes in expression of neuronal specific genes and alterations to development, while sparing the translation of shorter transcripts. [Speculation] […] Further studies using polysome and/or ribosome profiling should be able to rigorously test this hypothesis. [EXPLICIT CALL FOR RESEARCH]

✅ Clinical consequences of mammalian target of rapamycin complex 1 (mTORC1) upregulation may be amenable to tailored treatment with mTOR inhibitors. [Speculation] A clinical trial on an international scale would be needed to draw conclusions. [EXPLICIT CALL FOR RESEARCH]

✅ Thus, loss of function of Bassoon was potentially the underlying mechanism of the pathogenicity of BSN variants, which is consistent with the pathogenesis of other genes encoding CAZ proteins, such as UNC13A\n27 and UNC13B.6 [HYPOTHESIS] However, the functional effects of the BSN variants warrant further investigation.

✅ Existing medical research suggests that CUL3 mutations are closely related to neurodevelopmental disorder with or without autism or seizures (neurodevelopmental disorder with autism and seizures, OMIM: 619239). [Speculation] However, the number of published case reports of autism spectrum disorder due to CUL3 gene mutations is limited. [Suggests inadequate scientific support in available evidence]

✅ KV channel blockers may thus be of therapeutic interest for individuals affected with KCNA6-related epilepsy with associated gain-of-function mechanisms. [Speculation] However, to date and to our knowledge, no KV1.6\u2010 specific small molecule inhibitors of KV1.6 have been reported. [Indicates lack of supporting evidence]

✅ Adjunctive treatment with sirolimus or everolimus in the youngest patients with drug-resistant seizures could improve their epilepsy control, as was reported by a few studies [9, 27]. However, analyses were conducted only on a small number of patients. [Indicates lack of supporting evidence]

In the absence of clear “reasearch goal” markers → annotate as NOT goal for knowledge.

Examples:

❌ Interestingly, caspase-3 expression in the cortex was significantly lower in children with DRE compared to adults with epilepsy, whereas in the white matter it was higher in children. This suggests age-related differences in caspase-3 expression within the DRE patient cohort. However, we did not find significant differences in protein expression based on gender. These findings indicate that the level of caspase-3 expression may independently contribute to the pathogenesis of epilepsy and can vary with age.

❌ Regression analysis revealed that while GFAP levels were significantly elevated in both the cortex and white matter of epilepsy patients across all age groups, there was no statistically significant difference in GFAP expression between children and adults. The lack of age dependence suggests that the reactive production of GFAP by astrocytes is not specific to a particular age group and supports our hypothesis of a common pathogenesis of FCD-associated temporal lobe DRE in both children and adults.

❌ We found a statistically significant negative correlation between GFAP expression and disease duration in the white matter, while there were no statistically significant changes in the cortex, suggesting a limitation of reparative processes within the nervous tissue.

❌ Additionally, we found a positive correlation between S100\u03b2 protein expression in the white matter and both disease duration and caspase-3 protein expression. [FINDING] The increased presence of S100\u03b2 may potentially have proapoptotic effects, as it has been shown to upregulate nitric oxide (NO) expression, leading to neuronal and glial cell death and potentially participating in the pathogenesis of epilepsy [39].

❌ Notably, we found that all three disease-causing mutations increase the tRNA-binding capacity of eEF1A2. We hypothesize that the mutation-induced increase in tRNA binding results in a broad sequestration of all tRNAs and reduces tRNA availability, thereby reducing the rate of elongation.

❌ The evidence from ClinGen Framework suggested an association between BSN variants and epilepsy. Conclusion: The BSN gene was potentially a novel candidate gene for epilepsy. The phenotypical severity was associated with the genotypes and the molecular subregional effects of the variants.

❌ Therefore, our results suggest that the position of the identified GABRA1 variants may potentially predict the intensity of one or more of these clinical features, reflecting the severity of the condition. In this case, GABRA1 variants in the extracellular domain (L49H, P59L, W97R, D99G, G152S) will likely manifest with less severity, while variants in the transmembrane domain 1 (V270G), transmembrane domain 2 (T294R), and the linker between transmembrane domain 2 and 3 (P305L) may predict a more severe disease condition.

Exception #3

Statements that use ignorance terms such as “unknown” or “uncertain” as labels for classification, grouping or description rather than to indicate a scientific research target should NOT be annotated as Epistemic Gaps.

Examples:

❌ The aetiology of epilepsy is classified into genetic, structural, infectious, immunological, metabolic and unknown aetiologies.

❌ This study aims to explore the relationship between BSN variants and epilepsy. Methods: Whole-exome sequencing was performed in a cohort of 313 cases (trios) with epilepsies of unknown causes. Additional cases with BSN variants were collected from China Epilepsy Gene V.1.0 Matching Platform.

❌ In light of the paramount importance of GABAAR \u03b11 subunit and its variants described so far, the present study focused on one hundred thirty-seven non-synonymous single-nucleotide polymorphisms (nsSNPs) within the coding region of the \u03b11 subunit, which were categorized as variants of uncertain significance (VUS) in the ClinVar database.

❌ Addressing this, one hundred thirty-seven GABRA1 gene variants of unknown clinical significance have been extracted from the ClinVar database and computationally analyzed for pathogenicity.

❌ On the other hand, the number of variants with unknown clinical significance continuously increases with the availability of next-generation sequencing (NGS), a trend that is also critical for personalized medicine and pharmacogenomics.

❌ Thus, our data have important implications since predicting pathogenicity of specific genetic variants associated with epilepsy syndromes will contribute to variant interpretation for clinically unknown genetic variants, which provides important insights into the underlying causes of the condition and guiding treatment decisions, such as selecting targeted therapies.

❌ Thus, according to International League Against Epilepsy (ILAE), classification of the Epilepsies is a multi-level system including seizure types (focal onset, generalized onset and unknown onset), epilepsy types (Generalized, Focal, Combined Generalized and Focal, Unknown) and in the third level the epilepsy syndromes that are integrated with comorbidity and etiology which also includes genetic etiology.

❌ Combined with our study, a total of 88 patients with NPRL3-related epilepsy were found, of which 57 were boys and 30 were girls, respectively, while the gender of one case was unknown.

❌ Indeed, genetic testing has already become an integral part of the diagnosis for epilepsy, especially for unexplained epilepsy and pediatric epilepsy [41,42] and the phenotypic spectrum of GABRA1 gene such as infantile febrile seizures, bilateral\u2010tonic\u2010clonic seizures and atonic seizures has already been identified [32].

Non-Epistemic Gap

Non-Epistemic Gap identifies a gap or limitation in research or clinical practice with respect to available methods, technology, data, and therapeutic solutions. We identify four major categories of non-epistemic gap: methodological or technical gap, data gap, diagnostic gap and therapeutic gap.

Types of Non-Epistemic Gap

Methodological/Technical Gap refers to limitations in available methods or tools for research or clinical practice.

Examples:

❌ Computational methods developed to predict drugs for polygenic diseases are unsuitable for monogenic diseases in general and monogenic epilepsies in particular.

❌ Animal models of UBE3A overexpression have been used to study the syndrome, but with limited success in recapitulating the full range of Dup15q phenotypes.

❌ However, ES has been less powerful in detecting structural variations (SVs) that involve intronic, GC-rich or repetitive regions than detecting single-nucleotide variants (SNVs).

❌ Due to the limitation of WES, potentially disease-causing intron variants or copy number variants may not be detected, especially case 1 with only a heterozygous SZT2 variant.

❌ The generation of animal models for Dup15q has been difficult. […] However, this model has poor face validity as there were no behavioral phenotypes with maternal duplication, whereas paternal duplication resulted in subtle autism-like phenotypes (Nakatani et\u00a0al., 2009). […]
Nevertheless, the construct validity of this model came into question, as the C terminal of Ube3a was tagged with a FLAG tag that affected the catalytic function of the protein. […] Although these models might be valid for studying the cellular and behavioral effects of Ube3a overexpression, they fail to encompass the full range of severe Dup15q phenotypes and replicate the complex genetics of the disease.

❌Due to a size limitation, minigene constructs only contain intronic regions and neighboring exons, which may not recapitulate all the necessary splicing components, so results from a minigene assay only serve as a proxy for in vivo splicing changes.

❌ Furthermore, a causal relationship between c.791 + 6T>G and the observed splicing change in NUS1 cannot be formally established based only on in vivo results, because contributions from other genetic differences between the patient and her parents could not be ruled out. Therefore, minigene assay can help establish this causal connection by comparing the transcript products of two expression constructs that differ only at the variant site, although it takes time and effort to set up this assay.

❌ Last, mini mental and MOCA tests are limited tests to evaluate intellectual disability and are more reliable in patients older than 8 years and.

❌ Specifically, our results imply that the GoF versus LoF paradigm oversimplifies variant effect on channel function and does not fully capture the complete physiological impact of channel dysfunction.

❌ Our findings illustrate a prevalence of complex biophysical effects for many variants suggesting that a simple binary scheme to classify variants as either GoF or LoF is insufficient to capture the full spectrum of variant channel dysfunction.

Diagnostic Gap refers to limitations in available diagnostic solutions for clinical practice.

Examples:

❌ Due to overlapping phenotypes, among the patients, clinical subtyping was not possible. However, whole exome sequencing successfully pinpointed the molecular diagnosis which could be helpful for better management of these patients.

❌ Among the remaining patients with neuroimaging abnormalities, FCD was the common presentation of NPRL3-related epilepsy (62.5%), which was consistent with the previous studies (6). However, for those with negative brain MRI results, small FCD may be overlooked. Thus, a PET scan is needed in the future to reveal the subtle metabolic or biochemical function changes in the brain of patients with NPRL3-related epilepsy.

❌ However, despite the tremendous progress that has been made in the development of both sequencing technology and bioinformatics in recent years, it should be noted that other diagnostic tests are still necessary to confirm or reject a diagnosis with certainty in cases where this approach results in the identification of genetic variants of uncertain clinical significance.

❌ However, since it usually takes months to obtain genetic testing results, this has also limited clinical utility when deciding on anti-seizure medications at the time of seizure onset.

Therapeutic Gap highlights challenges and limitations in available therapeutic solutions.

Examples:

❌ The current treatment of epilepsy with NPRL3 gene variants is lacking in specificity.

❌ Although current anti-EP drugs can reduce the frequency of convulsive episodes, the disorder remains incurable [4].

❌ A drug that exerts a desirable effect in vitro, might not be able to penetrate the blood-brain barrier and produce a clinical antiseizure effect in vivo.

❌ Few licensed or experimental antiseizure drugs are efficacious in people with or animal models of Dravet syndrome.

❌ We aimed to create a computational method to predict the efficacy of drugs for monogenic epilepsies, and to use the method to predict drugs for Dravet syndrome, as (1) it is the archetypal monogenic catastrophic epilepsy; (2) few antiseizure medications are efficacious in Dravet syndrome;

❌ Complete seizure control remains unattainable for most people with the condition, and the goal of current therapy is to reduce the frequency of seizures while minimizing the adverse effects of drugs.

❌ With adequate serum concentration of antiepileptic drugs, the epilepsy symptoms in many patients still cannot be controlled well.

❌ Because of the poor control of the epilepsy symptoms in the refractory epilepsy group, clinicians are more inclined to increase the dosage and use multiple drugs for treatment.

❌ Although multiple antiepileptic drugs (AEDs) may be used alone or in combination, approximately one-third of people with epilepsy are unable to fully control their epilepsy, a phenomenon termed drug resistance (L\u00f6scher et al., 2020).

❌ Nevertheless, about 20% of epileptic patients, as defined as refractory epilepsy, fail to respond to ASMs [6].

❌ Overall, evidence robust enough to guide antiseizure medication (ASM) choices in genetic epilepsies remains limited to the more frequent conditions for which controlled trials and observational studies have been possible.

❌ Precision medicine remains applicable to only a narrow number of patients with monogenic epilepsies and may target only part of the actual functional defects.

Data Gap refers to unavailability of data to support scientific discovery.

Examples:

❌ For some drugs, the intermediate transcriptome score is not currently available, as their transcriptomic effects are still to be assayed.

❌ Brain tissue from people with monogenic epilepsies is scarce/unavailable.
(Focus is on scarcity of experimental material for clinical practice, no epistemic consequence discussed or need for research)

❌ Familial focal epilepsy with variable foci (FFEVF) is a rare type of focal epilepsy syndrome; it is associated with NPRL3 variant. However, relevant reports are rare in China.

❌ However, this is a rare condition, with limited reports in the literature.

❌ The VUS are so rare that there are no previous reports.

❌ Only twenty-four individuals with ST3GAL3-related congenital disorder of glycosylation (CDG) have been described in the literature.

❌ The MRI findings in our case were still present at the age of 5\u2009years, although follow-up imaging was not available for the two siblings in the literature.

❌ There were some missing data from literature review, and not all cases had all the reported variables of interest available.

❌ Nevertheless, for many variants, the functional testing in respective in vitro or in vivo systems is not available.

❌ Satterstrom et al. have reported six protein truncating variants (PTVs), four missense variants and two synonymous (15), whereas further phenotypic data were not specified.

❌ Few cases of patients with NEXMIF variants had been reported, and to date, no deaths have been reported to our knowledge.

❌ Other large cohorts of disease natural history didn’t present data on brain involvement [20].

❌ Other phenotypes, particularly those of mild/modDEE and self-limited familial infantile epilepsy, are relatively underrepresented.

❌ Loss-of-function variants have been less reported, and primarily cause neurodevelopmental delays with generalized epilepsy, neurodevelopmental disorder without epilepsy, intellectual disability, autism spectrum disorder, and mobility disorders.

Statements discussing non-epistemic limitations should be annotated as Goal for Knowledge only when the limitation is framed as a target for future research.

Examples:

✅ There is a need for dedicated enhanced computational methods to predict drugs for monogenic diseases, in general and monogenic epilepsies in particular.
(Acknowledges the need for technological innovation; Implied: is meant to be accomplished via scientific investigation)

✅ Our finding of mixed functional properties suggests a need for a more nuanced classification of SCN2A variant dysfunction.

✅ Our findings indicate that a more nuanced framework for describing and reporting the function effects of ion channel variants will be valuable.

✅ However, the explosion in genetic data makes the functional evaluation of individual variants using traditional whole-cell patch clamp techniques insufficient, and techniques with more scalable throughput are required to meet demand.

✅ As such, it is desirable to identify additional drugs that can be used to treat Dravet syndrome, so that any individual with the condition has more treatment options available and, hence, better chances of finding an efficacious and well-tolerated treatment.

Additional instructions for Therapeutic Gap annotation

A therapeutic gap statement qualifies as Epistemic Gap if it does at least one of the following:

Questions the effectiveness of therapeutic solutions implying the need for scientific confirmation

Expresses a lack of (knowledge about) effective therapeutic solutions

Calls for research to address a therapeutic gap (e.g., indicates the need for development and discovery of new drugs)

Examples:

✅ Specific Cav2.3 inhibitors would be required to test if inhibition of Cav2.3 could ameliorate CDKL5 deficiency phenotypes in mouse and human models.

✅ At present, no effective drugs have been found for this disease.

✅ There is no specific medicine for the NEXMIF variant, and it can only be treated according to the symptoms.

Study Limitations

Study Limitation statements qualify the validity of the current results, acknowledging sources of uncertainty or weaknesses in the design, methods, or analysis of the study that could affect the reliability, generalizability and interpretation of its findings (e.g., sample size, evaluation biases/inaccuracies, missing/unavailable data, etc.).

The following are examples of quality-oriented statements highlighting study-specific limitations:

❌ Here we should highlight that one of the limitations of our study is the brief period of follow-up of our patient, who is only at the age of 2 years at the time of writing.

❌ However, this study had several limitations. First, the sample size was small and we only investigated the roles of miR-30b-5p and GRIN2A in vitro and in vivo.

❌ The main limitation of this study is its retrospective character, which may hinder the objectivity and availability of all data. […] The seizure types and epileptiform discharge types could not be determined for some patients in the treatment and control groups because of missing data in the medical records. […] Secondly, the small cohort size, divided into smaller subgroups, could impede the statistical significance of the analysis.

❌ The Clinical Effect score might be biased against less commonly used and newer drugs, as there is less chance for their adverse effects to be reported and less time for the reports to accumulate.

❌ However, although differences are not significant due to insufficient patient numbers, epilepsy was present in 100% of patients (vs. 62% for other variants), unsupported walking was never achieved for 100% of patients (vs. 66%) and patients are non\u2010verbal (Table\u00a01).

❌ Moreover, other members of the family refused to provide blood samples due to privacy concerns. Therefore, only one unaffected family member was included in the genetic analysis. Consequently, it was difficult to calculate the penetrance accurately.

❌ Our work has limitations, including that this is a retrospective case series with few newly described cases. However, this is a rare condition, with limited reports in the literature. There were some missing data the from literature review, and not all cases had all the reported variables of interest available. [Data unavailability]

❌ Patients were likely to be misdiagnosed with sporadic epilepsy or other familial focal epilepsy forms until new affected members were identified, especially in smaller families. As the pedigrees were too small to demonstrate clear autosomal dominant inheritance, the prevalence of FFEVF was underestimated.

Annotate study limitation statements as Goals for Knowledge only when they highlight epistemic implications or indicate unfinished work, implying the need for additional work to address an unresolved research question.

Examples:

✅ “Our study has expanded the phenotype spectrum of FFEVF and found a new NPRL3 variant that had not been reported. Compared with unrelated healthy controls, we found that the NPRL3 variant was associated with the relative expression level of mRNA and abnormal splicing in vitro. However, the case-control design of the study involving unrelated healthy controls is highly underpowered for evaluating the pathogenicity of the variant.”

Epistemic Gap: variant pathogenicity is yet to be determined

✅ A detailed analysis of the relationship between gene variants and clinical phenotypes has not been conducted yet.

Epistemic Gap: missing genotype–phenotype analysis

✅ Epilepsy control after sirolimus and everolimus discontinuation has not been determined yet. Most patients in this study continued the treatment at the end of data collection, so seizure recurrence assessment in a long-term follow-up after mTORi cessation was impossible. The long-term effect of mTORi on epilepsy control after their discontinuation should be addressed in future studies.

Epistemic Gap: long-term effect of mTORi on epilepsy control is yet to be established

✅ This study has several limitations. The pathogenicity of the variants identified in this study was only supported by in silico analysis; the detailed functional alteration needs to be experimentally examined. Due to the limitation of WES, potentially disease-causing intron variants or copy number variants may not be detected, especially case 1 with only a heterozygous SZT2 variant. [Technical gap] The genotype-phenotype correlation needs more cases to further elucidate in the future. →

Epistemic Gap: functional validation of variants pathogenicity is missing

✅ Unfortunately, we could not explain the beneficial effect of VPA in several described patients by an effect of the drug on the KV3.2 channel. Again, more complex model systems such as iPSC-derived neurons from affected patients with appropriate expression of different ion channels may be needed to elucidate the underlying mechanism.

Epistemic Gap: explanation of the mechanism is missing

✅ One limitation of this study was the small number of patients in the ambulantory group, what could provide additional information about the gravity spectrum of the brain manifestations in LAMA2-RD [Goal for Knowledge]. Another limitation was the lack of neuropsychological evaluation and quality-of-life tests for a better clinical characterization of the patients. [Study Limitation] Last, mini mental and MOCA tests are limited tests to evaluate intellectual disability and are more reliable in patients older than 8 years.

Goal for Knowledge Gap: gravity spectrum of the brain manifestation in LAMA2-RD