A Study of the Clinical and Genetic Aspects of Familial Epilepsy

Ntenga P^1*, Touré Kamadore², Ilunga Kabulo M³, Koba Bora B¹, Alain Kasongo³, Numbi Kilumba S¹, Bugeme M¹, Mukeng KD³, Mulumba A⁴, Omangelo Shongo F¹, Sedar Ceylaner⁵, Muyumba K¹, Monga Ben⁶, Ngoy Nkulu D¹

¹University of Lubumbashi, Faculty of Medicine, DRC
²Cheikh Anta Diop University, Dakar, Senegal
³Neurodiagnostic Center of Lubumbashi, Lubumbashi, DRC
⁴Dr. Joseph Guislain Neuropsychiatric Center of Lubumbashi, Lubumbashi, DRC
⁵Genetics and Rare Diseases, Diagnosis Center, Ankara, Turkey
⁶University of Lubumbashi, School of Public Health, DRC, Senegal

*Correspondence author: Patrice Ntenga, University of Lubumbashi, Faculty of Medicine, DRC; Email: [email protected]

Citation: Ntenga P, et al. A Study of the Clinical and Genetic Aspects of Familial Epilepsy. J Neuro Onco Res. 2025;5(3):1-29.

Copyright^© 2025 by Ntenga P, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Table 1: Synthetic table of studies.

This study spanned a period from October 2020 to October 2024, which included epilepsy cases that presented for consultation during the study period. The case subject is the selected epileptic person. The exposure is the presence of a gene predisposing to epilepsy (reference gene). The control is a parent, brother, sister, first cousin or grandparent of the epileptic person. We reconstructed the family tree up to 3 generations:

• The first generation consists of the grandparents of the patient living with epilepsy
• The second generation consists of the parents of the patient living with epilepsy
• The third generation consists of the patient living with epilepsy, their brothers, sisters and first cousins. Thus, transmission is considered recessive when there is a skip of one generation without an epileptic patient and it is considered dominant when there is a case of epilepsy in each generation

Study Population

Patients aged 40 years or younger with epilepsy and their families received at the aforementioned investigation sites, with a notion of familial epilepsy discovered or reported during the consultation, were considered as potential candidates for this study.

Sample

We performed a stratified probabilistic sampling during this study for the selection of subjects who participated in it.

Inclusion Criteria:

• Patients aged 40 years or younger with epilepsy who signed the consent and/or assent to participate were considered in this study. This age was targeted because hereditary seizures tend to be expressed earlier
• However, we also included patients with late-onset seizures with a family history of epilepsy
• Be of the Black race and Congolese nationality (not from a mixed-race background)
• A cerebral CT scan was performed

Non-inclusion Criteria

Genetic pathologies in which epilepsy is not the essential symptom, such as Trisomy 21, were excluded from this study. Any epileptic or control subject with a hemoglobin level below 8 g% was also excluded (because this level could create a predisposition to generate a convulsive seizure and create a selection bias).

Data Collection Material

Data was collected and stored on data collection forms. At the end of each week, we systematically verified all the completed forms to ensure that they were properly filled out and to correct any errors the same day or the next day by re-consulting the files or registers. The family tree is established for the patient to see the recessive (if there is a generation skip) or dominant (disease present in each generation) nature.

Definition of Concepts

• Familial epilepsy: Family history of epilepsy
• Hereditary epilepsy: Epilepsy caused by genetic mutations, transmissible from parents to child
• Symptomatic epilepsy: Epilepsy secondary to a detectable brain lesion
• Sequencing: Procedure for determining the linear order of nucleotides of DNA in each cell of the organism
• Genome: The set of genetic information of an organism contained in each of the cells in the form of a chromosome
• Exome: The part of the genome that contains exons (the parts of genes that are expressed for protein synthesis)

Data Collection Method

The questions are open-ended. The patient themselves and/or the parents and/or companions of people living with epilepsy are subjected to the same questionnaire (see appendix). This was completed by a clinical examination, which will allow us to reduce selection bias.

Study Phases:

• Recruitment: Subjects were recruited by the principal investigator and his associates in the neurology departments of the Jason Sendwe Provincial General Reference Hospital, the Dr. Joseph Guislain Brothers of Charity Neuropsychiatric Center of Lubumbashi and the University Clinics of Lubumbashi. Other family members living with epilepsy were recruited through initial family contacts and/or their doctors. Our goal is to be as explicit as possible in the written and verbal consent procedures to ensure that all participants agree to join the study without excessive coercion. The principal investigator reserves the right to refuse any sample that, in his opinion, was given as a result of excessive coercion
• Pre-selection: Medical records supporting the diagnosis were taken and evaluated by the principal investigator and his associates to determine if the subject met the diagnostic criteria. The suitability for inclusion in the protocol was considered after reviewing the medical records. Subjects identified as meeting the diagnostic criteria were invited to come to the different follow-up appointments

Number of Visits and Subject Time Commitment

This protocol was initially implemented during the neurological consultation that was carried out at the aforementioned structures. Families were contacted by phone by a counselor or by the investigator for the collection of additional information on family history and access to the study.

Chronological Order of Study Procedures

Complete medical histories were gathered for each individual. This included information concerning pregnancy, childbirth, developmental stages and neurological disorders. Multigenerational family histories were obtained by establishing the family tree up to 3 generations. Invasive procedures were limited to blood collection (3.5 ml) using a 5 cc syringe with a fixed or winged needle. These samples were stored at -20°C with a laboratory refrigerator in 10 cc EDTA blood collection tubes after collection using P-10, P-100 micropipettes. The other selected controls were subjected to the same investigations only after they had completed the informed consent process. No therapeutic medication will be administered.

Laboratory Exome Sequencing Protocol

• Sequencing Location

The samples are genetically analyzed at the Intergen Genetics and Rare Diseases Diagnosis Research and Application Center Co. Ltd. Address: Mustafa Kemal Mah. 2119. Sokak No: 5 Çankaya/ANKARA Central Registry Number: 047801464 2900016 Tel: +90 312 428 48 14 – Fax: +90 312 428 26 93 – www.intergen.com.tr – [email protected] Document No: INT.KGE.FRM.21 24.08.2021 Rev.Date / No: –/00

• General Consideration

The analysis is performed to identify and characterize new epilepsy genes in Lubumbashi to explain the clinical (phenotypes) and genetic characteristics of the patients. These genes could probably be important in the normal function of the nervous system and also have important pathophysiological implications, as well as modifying the concept of epilepsy management not only in our community but also in other populations. Only observations with potential clinical significance for the phenotype are reported. Incidental findings are reported only if clinical action is necessary. Heterozygous variants of patients living with epilepsy are reported only at the request of the family. Only variants classified as pathogenic or likely pathogenic according to ACMG criteria are included in the report, whether they are incidental findings or heterozygous variants associated with epilepsy. Variants of Uncertain Significance (VUS) are reported only if they are relevant to the clinical indication. A Variant of Uncertain Significance (VUS) is on the verge of being classified as pathogenic if it is supported by evidence such as high scores on prediction tools, low frequency in the population, relevant bibliographic data and a strong clinical correlation with the preliminary diagnosis.

• Exome Sequencing

During the test, the DNA was first fragmented into small pieces, then enriched for exon and exon-intron junction regions, which are the most frequently altered regions of human DNA, allowing for the detection of monogenic diseases. DNA isolation was performed using standard French protocols. Genetic library preparation and exome enrichment were carried out using the TWIST human Comprehensive Exome kit. The enriched libraries were sequenced on the MGIT 7 instrument using PE150-FCL.12, 5 Gbp cartridges, which represents an average target coverage of 100 to 120x. The data were submitted to the GATK (Genome Analysis Toolkit) best practices workflow at the Intergen Genetics Center in Ankara, Turkey.

The raw reads were aligned to hg38 using bwa men 0.7.17. Then, the steps of duplicate marking and base recalibration were performed by GATK4. Variant calling was performed using two distinct algorithms, GATK UnifiedGenotyper and GATK HaplotypeCaller and both were complementary (Van Der Auwera et al. 2013). “GATK practices” refers to the “Best Practices” of GATK. These are bioinformatics protocols and pipelines carefully documented and optimized by the Broad Institute to maximize the accuracy and reproducibility of variant detection. These practices cover all steps, from the initial processing of raw sequencing data to obtaining a reliable list of genetic variants. Low-quality variants from two sets were eliminated based on read depth strand bias and quality parameters using the GAT SelectVariants option [35].

Key Stages Recommended by the Broad Institute

1. Raw Read Quality Control
   • Before any analysis, it’s crucial to ensure that the sequencing data are of good quality. Tools like FastQC are often used to evaluate reads (sequenced DNA fragments)
   • Sequencing adapters (short sequences added to DNA fragments) and low-quality bases are often trimmed or eliminated (for example, with tools like Cutadapt or Trimmomatic)

2. Read Alignment to the Reference Genome

• Cleaned reads are then aligned (mapped) to a reference genome (e.g., GRCh38 for humans)
• The result of this step is an alignment file (usually in BAM or CRAM format)

• BAM (Binary Alignment/Map) and CRAM (Compressed Reference-oriented Alignment Map) Formats
  • BAM is the compressed binary version of the human-readable SAM (Sequence Alignment/Map) format. It’s optimized for storage and fast computer access and its accompanying index file (.bai) allows for quick access to specific genomic regions. Each record in a BAM file contains detailed information about a sequenced read and its alignment, including the read’s sequence, base quality scores, alignment position and CIGAR string (which describes alignment operations like matches, insertions and deletions)
  • CRAM is a more recent evolution of BAM designed for even more efficient compression. Its main innovation is that it stores reads by recording only the differences between the read’s sequence and the reference genome, resulting in a significant reduction in file size (30% to 50% or more). CRAM can also be compressed with or without loss of information

3. Alignment Pre-processing

• This is a critical step of the GATK Best Practices to improve variant calling accuracy. It includes several sub-steps:
• Adding Read Groups: Ensures each read is associated with a read group containing important information (sample, library, etc.)
• Marking Duplicates: Identifies and marks reads from the same original DNA fragment (often due to PCR amplification). These duplicates are ignored during variant calling to avoid false positives
• Local Realignment around Indels: (Less critical with recent GATK versions) Corrects alignment errors at the edges of small insertions or deletions
• Base Quality Score Recalibration (BQSR): Corrects systematic errors in the quality scores assigned by the sequencer, thus improving the reliability of each base

4. Variant Calling
   • This is where GATK is most powerful. The primary tool for detecting germline (inherited) SNPs and Indels is GATK HaplotypeCaller
   • HaplotypeCaller reconstructs haplotypes (DNA sequences along a chromosome) and uses a probabilistic model to identify where variants are present and their genotype
   • Intergen also uses GATK GermlineCNVCaller to infer copy number variations (CNVs), which are deletions or duplications of larger genomic regions

5. Variant Filtering

• Raw variants called by GATK can contain false positives. Filters are applied to eliminate low-quality variants. Intergen mentions removing low-quality variants based on parameters such as strand bias, read depth and call quality

6. Functional Annotation of Variants

• Once high-quality variants are identified, they are annotated to understand their potential impact. Tools like ENSEMBL’s Variant Effect Predictor (VEP) are used to determine if a variant is synonymous, non-synonymous, truncating or if it affects a regulatory region
• Intergen also mentions comparing variants of interest with an in-house disease variant database

7. Visualization and Interpretation

• Variants of interest are often manually visualized using software like IGV (Integrative Genomics Viewer) to visually confirm the variant call

8. Variant Classification

• Variants are interpreted based on ACMG (American College of Medical Genetics and Genomics) criteria to assess their pathogenicity (whether they are the cause of a disease)

9. Final Annotation

• High-quality and significant variants are submitted for functional annotation using ENSEMBL’s Variant Effect Predictor [36]

10. Prioritization of Variants

• Rare variants (MAF < 1%) with a high impact, unknown significance, and/or potential splicing effects were prioritized. Other variants with potential effects on the observed phenotype were also analyzed

11. Visual Verification

• Variants of interest were visually verified on IGV and compared to an internal disease variant database by the Intergen Genetic Diagnosis Center in Ankara, Turkey [37]

12. Classification

• The classification of variants was performed taking into account the recommendations of the ACMG and ClinGen. It is important to consider that variant classification is based on current scientific knowledge and is progressive

Study Parameters

Sociodemographic Characteristics:

• Sex: male (M), female (F)
• Age (date of birth: day/month/year)

Clinical Parameters:

• Age of seizure onset (in years)
• Family history of epilepsy: brother/sister, parents, grandparent, paternal/maternal side.
• Clinical form of the epileptic seizure:
• Generalized seizure: GTC (Generalized Tonic-Clonic), atonic seizure, tonic seizure, myoclonic seizure, absence seizure
• Focal seizure: partial (without altered consciousness), complex (with altered consciousness)

Paraclinical Parameters:

• EEG: search for paroxysmal grapho-elements (spike-wave, polyspike, polyspike-wave, notched slow waves with synchronous spikes)
• Cerebral CT scan: search for the absence of a lesion in the cerebral parenchyma
• Exome sequencing: search for the type of mutations and/or variants in the selected epileptic patients and controls

Statistical Studies

Data were collected and entered using Epi InfoTM version 7.2.1.0 and SPSS software. Univariate analysis was used to calculate the frequency in terms of percentage for qualitative variables. The calculation of the mean, median and standard deviation was used for the analysis of quantitative variables. Bivariate analysis was used to compare the means according to the distribution of sociodemographic, clinical and paraclinical characteristics. The chi-squared test will be performed. The Odd Ratio (OR) with a 95% Confidence Interval (CI) and a significance was set for OR > 1 and a p < 0.01 to find the link between the variables.

Definition of the Disease, Exposure and Sequencing Procedure

• Case subject (the disease): the epileptic person selected for the study
• First exposed control: the parent of the selected patient
• Second exposed control: the brother or cousin of the patient, whether or not they have epilepsy
• Recessive transmission: characterized by a skip of one generation before an epileptic patient appears, according to the pedigree
• Dominant transmission: characterized by the presence of a case of epilepsy in each generation, according to the pedigree study
• Exome sequencing procedure:

• After collecting peripheral blood using P-10, P-100 micropipettes, these samples were stored at -20°C with a laboratory refrigerator in 10 cc EDTA blood collection tubes. In the laboratory, DNA was extracted for whole-exome sequencing

Ethical Considerations

The completion of this thesis, through the different stages carried out, respected the ethical principles guiding all scientific research. We took into account the four bioethical principles: autonomy, justice, non-maleficence and beneficence [38,39]. We obtained authorization from the Medical Ethics Committee of the University of Lubumbashi by letter referenced No: Approval: UNILU/CEM/2024/2018, issued on 01/23/2024. All study participants gave their informed consent to the survey team before the administration of the questionnaire and the collection of blood samples, either directly (adults) or indirectly (parents and/or companions of children). Participation in this study did not present any direct benefit for the patients living with epilepsy or their matched family members at the time of the study; however, the results stemming from this study could benefit other people in the same conditions. The principles of respect for research integrity guided the completion of this work through the different phases [40].

Study Limitations

Given that genetic analyses are expensive, it will not be possible to sequence all 72 patients to be matched with 2 other controls, for a total of 216 samples. Only 5 families are sequenced with their 2 matches total 15 samples, to present the first informative results; the others would only be sequenced if significant funding is obtained. Another limitation is the refusal by many families to allow blood to be drawn for analysis outside the country due to mystico-religious considerations.

Results

The results of the present study concern both the general characteristics of the sample and those of the family tree. The first part concerns the prevalence of familial epilepsy in Lubumbashi, the average age of patients and their sex.

1. General characteristics of the sample.

Prevalence

This Fig. 1 shows the percentage of familial epilepsy cases within the sample.

It appears from this figure that familial epilepsy is present at Lubumbashi in 11.23%.

Figure 1: Prevalence of familial epilepsy.

Age and Sex Table 1. Distribution of patients by mean age.

The mean age and median of patients with a history of familial epilepsy are shown in the following Table 2.

Table 2: Mean age of patients.

It appears from this table that the patients had a mean age of 15.24 ± 11.03 years, with a median of 14 years, 7 years at the 1^st quartile and 20 years at the 3^rd quartile. The distribution of patients according to their sex is shown in the Fig. 2 below.

It emerges from this Fig. 2 that the sex ratio is 1.32, in favor of men.

Figure 2: Distribution of patients by sex.

Distribution According to Other Parameters

Table 3-5 and Fig. 3-7 present other types of patient distribution.

Table 3: Distribution of patients according to seizure types.

We observe from this table that generalized tonic-clonic seizures were the most common with 70.83% of cases, followed by absence seizures with 8.33% of cases. The frequency of other seizures ranged between 1.39% and 4.17% of cases. The EEG results in patients with a family history of epilepsy are shown in Fig. 3 below.

1. Distribution of Patients According to EEG Results

Our results show that EEG was performed in the majority of patients (63.49% of cases) and was normal in 27.78% of cases and abnormal (presence of paroxysmal grapho-elements) in 35.71% of cases. The non-performance of this examination represented 38.57%.

Figure 3: According to EEG results.

Furthermore, the results of the patients’ therapeutic journey, shown in Fig. 4, indicate that in 48.61% of cases, patients had followed a drug treatment before arriving at the hospital and that traditional treatment (36.11%) and prayer (9.72%) accounted for a total proportion of 45.83%.

1. According to Mode of Transmission

In Figure 4, we considered the autosomal dominant and autosomal recessive modes.

We deduce from this figure that epilepsy is transmitted in the autosomal dominant mode in 41.67% of cases and in the autosomal recessive mode in 33.33% of cases.

Figure 4: Distribution of patients according to the mode of transmission by pedigree.

1. According to Consanguinity Between Parents

The results regarding the notion of consanguinity between the parents of patients living with epilepsy are shown in Fig. 5-7, indicating that no patient resulted from a consanguineous marriage.

Figure 5: Distribution of patients according to the notion of consanguinity between the parents of patients living with epilepsy.

Distribution of patients according to two associated parameters.

Table 4: Distribution of patients according to sex and seizure type.

Reading this table shows us that generalized tonic-clonic seizures are present in considerable proportion in both sexes (64.5% of cases for females and 75.6% of cases for males) followed by absence seizures in males with 9.76% of cases, for a corrected chi-square of 6.1.

According to Mode of Transmission and Seizure Type

Table 5: Distribution of patients according to mode of transmission and seizure type.

It appears from this table that CGTC are transmitted in 70% of cases according to the autosomal dominant mode and in 62.5% of cases according to the autosomal recessive mode, while absence seizures are transmitted in 12.5% of cases according to the autosomal recessive mode, with a corrected chi-square of 11.46.

Sequencing Results

Presentation of Sequenced Cases Based on Pedigree and Exome Sequencing Results (Fig.5,6)

Figure 5: Presentation of Sequenced Cases Based on Pedigree.

Figure 6: Presentation of exome sequencing.

Sequencing Result

Presentation: Sequencing Information Sheet for Family 1

Age of seizure onset: 2 years.

Seizure type: CGTC (Generalized Tonic-Clonic Seizure)

Therapeutic protocol: Monotherapy: DPK

Pedigree 3: Presentation of sequenced Family 1.

Sequencing Result

Presentation: Sequencing Information Sheet for Family 2

Age of seizure onset: 2 years

Seizure type: ABSENCE

Therapeutic protocol: Bitherapy: DPK + KEPPRA

Pedigree 4: Presentation of sequenced Family 3.

Sequencing Result

Presentation: Sequencing Information Sheet for Family 2

2A: Sex: F, 18 years old, student

Age of seizure onset: 9 years

Seizure type: ABSENCE

Therapeutic protocol: Bitherapy: DPK

Sequencing Result

Presentation: Sequencing Information Sheet for Family 4

In Table 6,7, we present the results of the sequenced families in the first and those evaluating the clinical validity of specific gene-disease associations (concordance) ACMG in the second, respectively.

Table 6: Sequencing results by family.

Table 7: Presentation of results based on phenotype (clinical) – genetic concordance.

Discussion and Description of the Genes Found

General characteristics of the sample.

• Prevalence of Familial Epilepsies

Our study collected 72 families with a history of familial epilepsy out of a total of 641 patients diagnosed with epilepsy, representing a prevalence of 11.23%. This result is consistent with findings from other authors, such as M. Ndiaye et al. in Senegal, who reported a general frequency of familial epilepsy at 11%.

A similar finding was made by Kuate-Tegueu Callixte in Cameroon in 2014, who found that over half (57.1%) of patients living with epilepsy had a family history of the condition. Of these, 11.4% had two family members with epilepsy and 10% had three or more family members affected by the disease [41].

• Notion of Consanguinity

Lamine Thiam in Senegal (2020), in his study on the clinical and paraclinical aspects of idiopathic childhood epilepsy, showed that out of 18 children followed for idiopathic epilepsy, parental consanguinity and family history of epilepsy were found in 1 and 6 children, respectively (5.5% and 33.3%) [42]. Our study’s prevalence of 11.23% without any notion of parental consanguinity (Fig. 5) suggests an inherited nature that needs to be demonstrated. For many years, the role of ion channels in epilepsy has been suspected based on physiological arguments, as ion channels (Na+, K+) are responsible for generating action potentials and many anti-epileptic drugs act directly or indirectly on their function. Recent molecular genetics data have confirmed this role, with studies of familial epilepsy forms implicating genes that code for subunits of ion channels or receptor channels [43]. In their studies on epilepsy, a family history of the disease was found to be 2 to 3 times more common in patients with epilepsy than in controls [44-46]. Literature has shown that consanguineous marriages are common in some cultures in Asia, particularly in Indian or Muslim communities. In one study in India, consanguineous parents were more frequent among patients than controls (13.1% vs. 6.6%) (2.6; 95% CI: 1.5-4.4). The association with consanguinity is stronger in generalized epilepsies than in localized ones and in idiopathic or cryptogenic epilepsies than in symptomatic ones [45,47].

• Average Age and Sex

As shown in Table 2 and Fig. 2, the average age at diagnosis for patients with epilepsy in our cohort is 15.24±11.03 years, with a maximum of 20 years for 75% of cases and a mode of 17 years. Zubcevic reported an average age at diagnosis of 5 years. Many other studies report a high frequency of seizures with a hereditary character in children [48-50]. In their studies, Dadah Samy Mohamed Lemine, et al., in Senegal and Mukuku O, et al., in the DR Congo reported an average age of patients with epilepsy around 8 years in Senegal, with a peak in the 5 to 10-year age group [51]. A male predominance was also observed in our study with a sex ratio of 1.32. This male predominance could be explained by a possible neurobiological difference between the neurons of male and female subjects, leading to different responses to brain injuries [51]. This male overrepresentation could also be explained in our communities by the underreporting of the disease in young women of marriageable age. Amina Chentouf, et al., in Algeria found a sex ratio of 1.35 in favor of the male sex in their study on the clinical characteristics and hereditary profiles of epilepsies, with no mention of X-linked transmission. [20].

• Type of Seizures

We found that generalized tonic-clonic seizures were the most common, accounting for 70.83% of cases, followed by absence seizures with 8.33% of cases. The frequency of other seizures ranged from 1.39% to 4.17%. In his study on epilepsy and schooling in Congolese epileptic children, Ntenga Parice, et al., [29]. found that generalized tonic-clonic seizures were the most frequent, with 41.7% and a p-value of 0.04, followed by absence seizures with 33.3% and a p-value of 0.67. The high representation of generalized tonic-clonic seizures could be justified by their dramatic and easily recognizable nature, as well as by the population’s lack of awareness of other non-convulsive seizure manifestations. This same finding has been made in most studies conducted in Africa [51,20].

Genetic Section

1. Mode of Transmission of the Pathology According to the Pedigree

We observed from Figure 4 that the autosomal dominant mode of transmission accounts for 41.67% of cases, while the autosomal recessive mode of transmission accounts for 33.33% of cases. This result contradicts that of MEFOUNG Ephrata Samuel (2021) [52], for whom the autosomal recessive mode of transmission represented more than half of the cases. However, our results are consistent with a study conducted in Algeria (2015) by Chentouf A, et al., who found a high prevalence in favor of the autosomal dominant mode [20].

These differences could be justified by the different sample sizes, which are not large enough to characterize a distribution on a national scale and determine the predominance of one mode of transmission over another.

2. Description of the Genes

Gene ATP2B1 [53-55]

1. Definition and Nature of the ATP2B1 Gene: NM_001366521.1 c.3356G>A (p.Arg1119Gln)

The ATP2B1 gene codes for PMCA1 (Plasma Membrane Calcium ATPase 1), a calcium pump located in the plasma membrane of our cells. Its main physiological role is to maintain intracellular calcium homeostasis, precisely regulating the concentration of calcium inside cells.

• Understanding the Gene Nomenclature

• Gene ATP2B1: The gene’s name, standing for “ATPase plasma membrane Ca2+ transporting 1”. It codes for the PMCA1 protein.
• 1: The accession number of the reference transcript (mRNA) for this gene in the NCBI RefSeq database. The “.1” indicates the version of the transcript, which is crucial for unambiguously describing mutations.
  • Interpretation of the Mutation: c.3356G>A (p.Arg1119Gln)
• 3356G> A: This refers to the coding DNA sequence (cDNA). The mutation is at the 3356th nucleotide, where a Guanine (G) has been replaced by an Adenine (A).
• (Arg1119Gln): This refers to the protein sequence. The change in the DNA leads to the replacement of the amino acid Arginine (Arg) at position 1119 with Glutamine (Gln). This is a missense mutation.

2. Key Physiological Functions of the ATP2B1 Gene [53-55].
   • Calcium Export from the Cell: PMCA1 actively pumps calcium ions (Ca²⁺) out of the cell, which is crucial as high intracellular calcium can be toxic and interfere with many cellular functions
   • Blood Pressure Regulation: ATP2B1 plays a role in regulating blood pressure by modulating the contraction of vascular smooth muscle cells.
   • General Cellular Calcium Homeostasis: PMCA1 is ubiquitously expressed, highlighting its fundamental role in maintaining calcium levels in almost all eukaryotic cells.
   • Calcium Signaling: By controlling calcium levels, ATP2B1 indirectly participates in many calcium-dependent cellular signaling pathways.
   • Brain Development and Neuronal Function: Mutations in the ATP2B1 gene have been associated with neurodevelopmental delays, mild intellectual disability and language delays, suggesting its involvement in brain function.
   • Insulin Sensitivity Regulation: Research indicates that ATP2B1 may influence insulin sensitivity by regulating the calcium/calmodulin signaling pathway.
   • Osteoclast Development and Survival: It plays a dual role in the differentiation and survival of osteoclasts (cells involved in bone resorption) by regulating calcium oscillations.

3. Consequence of the Mutation in the ATP2B1 Gene

This is a missense mutation, resulting in a single amino acid change. The severity of the impact depends on the nature and position of the amino acid. The replacement of the basic, polar Arginine with a polar but uncharged Glutamine could potentially alter the structure or function of the protein. The location of Arginine at position 1119 is in an important region of the PMCA1 protein. Studies have shown that missense mutations in this gene, especially those affecting key functional domains, can significantly decrease the calcium pump’s ability to export calcium from the cell. The neurological consequences of ATP2B1 mutations are attributed to the disruption of intracellular calcium regulation in neuronal cells. Calcium is a vital secondary messenger involved in many neuronal processes. The main neurological consequences are:

• Global Developmental Delay (GDD) and Intellectual Disability (ID): The most frequently reported consequence, with delays in acquiring key developmental milestones.
• Language Delay: Speech and language development are often affected.
• Motor Delay: Motor skills, such as walking, can be delayed.
• Epileptic Seizures (convulsions): A significant percentage of individuals with ATP2B1 mutations experience seizures of various forms.
• Rarity of Specific Monogenic Mutations: Mutations in single genes like ATP2B1 are generally rare in the general population with epilepsy. However, the clinical and research community recognizes ATP2B1 as a relevant gene for epilepsy in syndromic contexts.
• Underlying Mechanisms of Seizures: The neurological consequences are attributed to the disruption of calcium regulation, which is essential for neurotransmitter release, synaptic plasticity, neuronal development and excitability. Unregulated excess of intracellular calcium can lead to neuronal hyperexcitability, potentially contributing to seizures.
• Hypotonia: A decrease in muscle tone.
• Brain MRI Abnormalities: Structural brain alterations have been reported in some individuals.
• Behavioral Disorders: Abnormal behaviors have been described.
• Ataxia (less frequently): Problems with movement coordination.

 Discussion of the ATP2B1 Gene [55,56].

Frequency and Phenotypes of Mutations in Epilepsy

Mutations in the ATP2B1 gene are a very rare cause of epilepsy. In contrast to genes like SCN1A or KCNQ2/3, which are mutated in a significant proportion of cases (for example, SCN1A is responsible for 35% of Dravet Syndrome cases), the contribution of ATP2B1 is minimal. This explains why it is often associated with rare neurodevelopmental syndromes. A 2022 study identified de novo variants of ATP2B1 in a cohort of 12 individuals with neurodevelopmental disorders, among whom 6 individuals (50%) presented with epileptic seizures of different forms [55].

Gene CACNA11

Physiology to Pathology of the Identified Gene

1. General Characteristics of the Gene: CACNA11; NM_021096.4 c.2059A>G p. (Met687Val)

• Gene: CACNA11 (“Calcium Voltage-Gated Channel Auxiliary Subunit Alpha 11”).
• Variant Name: NM_021096.4 c.2059A>G p. (Met687Val).
• 2059A>G: Adenine (A) at position 2059 is replaced by a Guanine (G) in the DNA.
• (Met687Val): The amino acid Methionine (Met) at position 687 is replaced by Valine (Val) in the protein.
• Zygosity: Heterozygous. The person has one mutated copy and one normal copy of the gene.
• Heredity: Autosomal dominant. A single mutated copy can be sufficient to cause the disease. This inheritance pattern often shows variable age of onset, incomplete penetrance and differential expression, meaning that some family members with the variant may be unaffected or have partial symptoms. Family screening is recommended to clarify the clinical significance of the variant.

Physiological Roles of the CACNA11 Gene

• Formation of Voltage-Gated Calcium Channels: CACNA11 provides instructions to create a subunit of voltage-gated calcium channels. These channels are proteins that form pores in cell membranes, allowing calcium ions to enter.
• Regulation of Calcium Influx: Calcium channels are crucial for regulating calcium entry into cells in response to changes in the cell’s membrane potential, especially in the nervous system.
• Cell Communication and Neuronal Functions: Calcium influx is vital for nerve cell communication, neurotransmitter release, muscle contraction and gene expression regulation.

• In summary, the CACNA11 gene is fundamental for controlling calcium entry into cells, which has a direct impact on electrical signaling and cellular functions, particularly in the brain and nervous system [57, 58].

Consequence of the Mutation in the CACNA11 Gene

A mutation in the CACNA11 gene can lead to a neurodevelopmental disorder with speech problems and with or without seizures. According to a recent study, 87% of patients with CACNA1A gene mutations were diagnosed with epilepsy and these mutations could lead to a wide range of seizure phenotypes.

Mechanisms Underlying Epilepsy in Case of Mutation

The CACNA1H gene codes for the alpha-1H subunit of the T-type calcium channel (also called Cav3.2). These channels play a crucial role in neuronal excitability, particularly in the thalamus, a brain region involved in generating cortical rhythms and seizures, especially absence seizures.

Mechanisms Underlying Epilepsy in Case of CACNA1H Mutation c.2059A>G p. (Met687Val)

The specific mutation is a missense mutation that leads to the substitution of Methionine with Valine at position 687 of the Cav3.2 protein. The mechanisms by which “gain of function” mutations like this one contribute to epilepsy are mainly related to an increase in neuronal excitability:

• Increased T-type Calcium Current (Gain of Function): The mutation can shift the activation curve to more negative potentials, causing the channel to open more easily or slow down inactivation, prolonging the channel’s opening. This leads to neuronal hyperexcitability, promoting the generation and propagation of epileptic discharges.
• Impact on Thalamocortical Oscillations:2 channels are particularly important for generating normal and pathological rhythmic oscillations (like the 3 Hz spike-and-wave discharges of absence seizures). Hyperactivity of these channels can disrupt these oscillations, transforming them into synchronous discharges that spread to the cortex, causing epileptic seizures.
• Altered Neuronal Electrical Properties: The mutation can directly affect the intrinsic properties of neurons, making them more prone to paroxysmal discharges.
• Indirect Change in Gene Expression: Some research suggests that increased Cav3.2 activity can influence calcium-regulated transcription factors, modifying the expression of genes involved in neuronal development or synaptic plasticity, which could contribute to a long-term epilepsy-prone environment [59,60].

Discussion of the CACNA1A gene [61,62].

Frequency and Phenotypes of Mutations in Epilepsy

The CACNA1A gene mutation is a rare cause of epilepsy. Its frequency in the general epileptic patient population is very low. Available data primarily comes from cohorts of patients with rare epilepsies or epileptic encephalopathies, where the percentage is higher.Furthermore, the rarity of these mutations contrasts with that of genes more commonly associated with epilepsy, such as SCN1A (linked to Dravet syndrome), where the mutation rate can reach 35% in familial cases.

Gene SCN3A; NM_006922.4 c.5164_5166delinsAAG p. (Ala1722Lys)

• Physiology to Pathology of the Identified Gene [63, 64, 65].

1. General Characteristics of the Gene:

• Gene SCN3A: Provides instructions for a protein subunit that is part of a sodium channel. These channels are essential for the normal function of nerve and muscle cells.
• 4: The accession number of the reference mRNA sequence for the SCN3A gene.
• 5164_5166delinsAAG: This is the DNA variant nomenclature. It means the nucleotides at positions 5164, 5165 and 5166 were deleted and a new sequence of nucleotides (AAG) was inserted in their place.
• (Ala1722Lys): This is the protein variant nomenclature. The amino acid Alanine (Ala) at position 1722 has been replaced by Lysine (Lys).

2. Clinical Significance: The variant is classified as a “Strong Variant of Uncertain Significance (SVUS).”
3. Family Implications: The patient and their sister are heterozygous for this variant, while their father is normal.

• Physiological Roles of the SCN3A Gene (Sodium Voltage-Gated Channel Alpha Subunit 3). The SCN3A gene codes for the alpha subunit of the Nav1.3 sodium channel.

These channels are crucial for generating and propagating action potentials in neurons and muscle cells.

• Expression and Role in Fetal Brain: Nav1.3 is highly expressed in the fetal brain and plays an important role in the development and folding of the cerebral cortex (gyrification).
• Neuronal Electrical Activity: These channels allow positively charged sodium ions to enter brain cells, activating neurons and enabling the transport of electrical signals.
• Role in Neuronal Plasticity: Although its expression is low in the normal adult brain, Nav1.3 can be upregulated in response to nervous system injuries, suggesting a role in post-injury neuronal plasticity.
• Involvement in Neuropathic Pain: Nav1.3 also induces increased excitability of sensory neurons, a lower nociceptive threshold and neuropathic pain
• Consequence of the Mutation in the SCN3A Gene [66,64].

Mutations in the SCN3A gene are responsible for a range of neurological conditions called SCN3A-related neurodevelopmental disorders (SCN3A-NDD). These complications can vary in severity:

• Severe and Early-Onset Epilepsies:
• Developmental and Epileptic Encephalopathy (DEE): Characterized by intractable seizures with developmental delays.
• Familial Focal Epilepsy with Variable Foci: Milder forms may include focal epilepsy with or without brain malformations.
• Most pathogenic variants (about 91%) lead to a gain of function of the channel, causing increased sodium channel activity and neuronal hyperexcitability.
  • Malformations of Cortical Development (MCD): More than 75% of individuals with SCN3A-related disorders have brain malformations, often bilateral diffuse or perisylvian polymicrogyria (multiple small convolutions creating excessive folding of the cerebral cortex).
  • Developmental Delays and Intellectual Disability: All affected children show some degree of developmental delay, which can include speech/language delay and intellectual disability.
  • Oral Motor and Speech Disorders: Difficulties with speech, swallowing and tongue movements.
  • Autonomic Dysfunctions: Episodes of asymmetrical facial flushing, excessive sweating, apnea and bradycardia.

Gene G6PD; NM_001360016.2 c.202G>A p. (Val68Met) [67-70].

1. General Information
   • G6PD gene: This is the gene for Glucose-6-Phosphate Dehydrogenase (G6PD). This enzyme plays a crucial role in red blood cell metabolism, protecting them from oxidative stress.
   • 2: This is the accession number for the reference mRNA sequence of the G6PD gene.
   • 202G>A: This is the nomenclature of the variant at the DNA level (coding sequence). The c. indicates that the variation is described at the coding sequence level. The 202G>A means that the Guanine (G) nucleotide at position 202 in the DNA sequence has been replaced by Adenine (A).
   • (Val68Met): This is the nomenclature of the variant at the protein level. The p. indicates that the variation is described at the protein level. Val68Met means that the amino acid Valine (Val) at position 68 of the protein has been replaced by the amino acid Methionine (Met) due to the DNA change.
2. Clinical Significance

This variant is classified as Pathogenic with low penetrance. This indicates strong evidence that this variant is a direct cause of a disease.

• Inheritance: X-linked. This means the gene is located on the X chromosome.
• In females (who have two X chromosomes), if only one copy carries the variant, they may be carriers and have milder symptoms or be asymptomatic due to X-inactivation.
• In males (who have only one X chromosome), if their single X chromosome carries the variant, they will generally develop a more severe form of the disease.

3. Neurological Consequences of the G6PD Gene Mutation;2 c.202G>A p. (Val68Met) [71,72].

Although the most well-known consequences of G6PD deficiency are hematological (hemolytic anemia, neonatal jaundice), neurological complications can occur, particularly in severe cases or specific contexts.

• Kernicterus (Bilirubin Encephalopathy): This is arguably the most severe neurological complication, primarily seen in newborns with G6PD deficiency. Severe, untreated neonatal jaundice can lead to the accumulation of toxic levels of bilirubin in the brain, causing irreversible brain damage. Symptoms can include lethargy, poor feeding, fever, high-pitched crying, changes in muscle tone and seizures. This can result in lifelong disabilities.
• Cerebral Oxidative Stress: G6PD is essential for the production of NADPH, a key component of cellular defense against reactive oxygen species (ROS). A G6PD deficiency can lead to increased oxidative stress not only in red blood cells but potentially in other tissues, including the brain. Although less directly studied for the specific c.202G>A p. (Val68Met) mutation, a general G6PD deficiency can impair the ability of brain and nerve cells to fight oxidative stress, potentially contributing to neurodegeneration and affecting neuronal survival.
• Altered G6PD activity in the brain could lead to high oxidative stress, affecting calcium mobilization, neuronal apoptosis, ion transport and excitotoxicity, thus impacting brain homeostasis.

4. Implications:

• Since there is a possibility of mild symptoms in female carriers due to X-inactivation, clinical evaluation of the individual is recommended.
• There is a 50% risk for the carrier to transmit this variant to their offspring.

The NPRL2 Gene, NM_006545.5 c171-9 A >G (IVS2-9 A>G) [73-75]

Definition: The NPRL2 gene (for “Nitrogen Permease Regulator-Like 2”) is an important gene in the human body.

Physiology

• Function: The NPRL2 gene provides instructions to make a protein that is part of a protein complex called GATOR1.
• GATOR1 Complex: This complex plays a crucial role in regulating a cellular signaling pathway called the mTOR pathway (mammalian Target Of Rapamycin).
• mTOR Pathway Role: The mTOR pathway is fundamental for many cellular processes, including:

• Cell growth and division
• Metabolism
• Protein production
• Autophagy (the process of recycling cellular components)
• In the brain, it regulates the development and adaptability (plasticity) of nerve cells.
  • GATOR1 Regulation on mTOR: The GATOR1 complex acts as a negative regulator of the mTORC1 pathway (a sub-complex of mTOR), especially when amino acid levels in the cell are low. In other words, it helps to “brake” mTORC1 activity.
  • Clinical Implication: Mutations in the NPRL2 gene can lead to a dysfunction of the GATOR1 complex, which causes hyperactivity of the mTOR pathway. This hyperactivity is strongly associated with certain diseases, including:
• Familial Focal Epilepsy with Variable Foci (FFEVF): This is the most known association. Hyperactivation of the mTOR pathway in the brain can cause changes in neuronal connections and increased excitability of these cells, which can trigger seizures.
• Potential role as a tumor suppressor: Studies suggest that NPRL2 may also have a tumor suppressor function and decreased NPRL2 expression has been associated with some cancers.

Meaning of the Variation NM_006545.5 c.171-9 A > G (IVS2-9 A>G): This variation signifies a change from an adenine (A) to a guanine (G) at the 9th nucleotide position upstream of the beginning of exon 2 (or intron 2) of the NPRL2 gene. This type of intronic variation is particularly important because it can affect RNA splicing.

• 171-9 A > G: This is the standard HGVS (Human Genome Variation Society) nomenclature for describing a variation in the complementary DNA (cDNA), which corresponds to mRNA. The c. indicates cDNA. The 171-9 indicates the position, with -9 meaning the variation is located 9 nucleotides upstream of position 171 within the intron before the exon. A > G indicates the base change.
• IVS2-9 A>G: This is an older, but still commonly used, notation that confirms the location. IVS2 means “Intron 2 Splice site,” and -9 confirms the position.
• Consequences of Splicing Variation: A variation in a splicing site can lead to exon skipping, intron retention or the activation of a cryptic splice site. These modifications can alter the protein’s sequence, make it non-functional or lead to its premature degradation, which can have pathological consequences.

Underlying Mechanisms of Epilepsy in the Case of an NPRL2 Mutation [76-78]

The NPRL2 gene is a subunit of the GATOR1 protein complex, which is a crucial negative regulator of the mTORC1 signaling pathway.

1. GATOR1 Loss-of-Function and mTORC1 Hyperactivation:

• Pathogenic mutations in NPRL2 (and other GATOR1 complex genes like DEPDC5 and NPRL3) lead to a loss of function of the GATOR1 complex.
• The normal role of GATOR1 is to brake mTORC1 activity.
• When GATOR1 function is lost due to an NPRL2 mutation, this brake is released, leading to abnormal and constitutive hyperactivation of the mTORC1 pathway.
• The c.171-9 A > G splicing variant can disrupt normal mRNA splicing, leading to a truncated, unstable or non-functional NPRL2 protein.

2. Consequences of mTORC1 Hyperactivation in the Brain:

• Focal Cortical Dysplasia (FCD): Although many GATOR1-related epilepsies are non-lesional, FCD (a cortical malformation associated with severe, often drug-resistant epilepsy) has been reported. mTORC1 hyperactivity is known to disrupt neuronal migration, cell proliferation and synapse formation, which can lead to architectural brain abnormalities.
• Neuronal Hyperexcitability and Excitation/Inhibition Imbalance:
• Increased Intrinsic Neuronal Excitability: mTORC1 activation can influence the expression of ion channels, such as voltage-gated sodium channels (e.g., Scn1A), leading to increased strength of neuronal action potentials and hyperexcitability.
• Synaptic Alterations: Hyperactivity can cause aberrant connections between neurons or an imbalance between excitatory and inhibitory signals, promoting seizure propagation.
• GABAergic Network Dysfunction: Studies in animal models show that GATOR1 loss of function can reduce the density of inhibitory GABAergic neurons’ dendrites, decreasing inhibition and increasing network excitability.
• Amino Acid Metabolism Abnormalities: mTORC1 is sensitive to amino acids. NPRL2 mutations can alter amino acid homeostasis in the brain. Some recent studies suggest an increase in glycine, a neurotransmitter, which may contribute to synaptic dysfunction and epilepsy.
• Increased Neuronal Size: mTORC1 hyperactivation is associated with increased cell growth, which can manifest as dysmorphic and hypertrophied neurons, typical of “mTORopathies.”

Therapeutic Options

Since mTORC1 pathway hyperactivation is the key mechanism of epileptogenesis in GATORopathies, mTOR inhibitors are the most promising therapeutic target.

• Rapamycin (Sirolimus) and its analogs (Everolimus):

• These are well-known mTOR inhibitors already approved for other “mTORopathies” like Tuberous Sclerosis Complex (TSC), where they have shown a significant reduction in seizure frequency.
• Mechanism: They bind to the FKBP12 protein and this complex then binds to and inhibits mTORC1 activity.
• Application in NPRL2-related epilepsy: Preclinical studies on animal models have shown that rapamycin can significantly reduce seizure frequency.
  • Gene Therapies and Nucleic Acid-Based Approaches:
• These are largely in the preclinical research stage but represent the future of precision medicine for genetic epilepsies.
• Gene Replacement/Correction Therapy: The idea is to introduce a functional copy of the NPRL2 gene or correct the existing mutation to restore normal GATOR1 function.
• Antisense Oligonucleotides (ASOs): For a splicing variant like c.171-9 A > G, ASO strategies could be used to correct the abnormal splicing. Success has been achieved with other neurological diseases (like spinal muscular atrophy with nusinersen).
  • Ketogenic Diet: This high-fat, low-carbohydrate diet is known for its effectiveness in certain drug-resistant epilepsies. It may have a moderating effect on the mTOR pathway, though the exact mechanisms are not fully understood in this specific context.
  • Epilepsy Surgery: For patients with identifiable and localized structural brain abnormalities (like FCD), epilepsy surgery can be an option, especially for those refractory to conventional treatments.

Conclusion

Our study of the clinical and genetic aspects of familial epilepsy revealed that:

• The prevalence of familial epilepsy in Lubumbashi is 11.23%
• Generalized tonic-clonic seizures and absence seizures are the most frequent in Lubumbashi, with 70.83% and 8.33% of cases, respectively
• The discovery of other genetic variants/mutations related to epilepsy in Lubumbashi after whole-exome sequencing highlights the difficulties in managing patients with epilepsy
• The genetic variants/mutations found in cases of idiopathic generalized tonic-clonic seizures in Lubumbashi are ATP2B1 and SCN3A, while those associated with absence epileptic seizures are CACNA1I and NPRL2
• These findings should be shared with chemists to open a path for the synthesis of targeted medications

Recommendations

• To patients with epilepsy and their families: Consult with experts for rational management of epilepsy
• To political and administrative authorities: Make epilepsy a public health priority to encourage investment in research and enable the synthesis of medications for targeted epilepsy management

Conflict of Interest

The investigators declare no material or financial conflict of interest related to this study.

Funding Sources

The completion of this thesis was made possible thanks to the support of the Intergen Genetic Diagnosis Center in Ankara, Turkey and the Neurodiagnostic Center of Lubumbashi.

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