A de novo pathogenic variant in MICAL‐1 causes epilepsy with auditory features

Abstract

Familial epilepsy with auditory features (FEAF), previously known as autosomal‐dominant lateral temporal lobe epilepsy (ADLTE) is a genetically heterogeneous syndrome, clinically characterized by focal seizures with prominent auditory symptoms. It is inherited with autosomal‐dominant pattern with reduced penetrance (about 70%). Sporadic epilepsy with auditory features cases are more frequent and clinically indistinguishable from familial cases. One causal gene, MICAL‐1, encodes MICAL‐1, an intracellular multi‐domain enzyme that is an important regulator of filamentous actin (F‐actin) structures. Pathogenic variants in MICAL‐1 account for approximately 7% of FEAF families. Here, we describe a de novo MICAL‐1 pathogenic variant, p.Arg915Cys, in a sporadic case, an affected 21‐year‐old Italian man with no family history of epilepsy. Genetic testing was performed in the patient and his parents, using a next‐generation sequencing panel. In cell‐based assay, this variant significantly increased MICAL‐1 oxidoreductase activity, which likely resulted in dysregulation of F‐actin organization. This finding provides further support for a gain‐of‐function effect underlying MICAL‐1‐mediated epilepsy pathogenesis, as previously seen with other pathogenic variants. Furthermore, the case study provides evidence that de novo MICAL‐1 pathogenic variants can occur in sporadic cases with epilepsy with auditory feature (EAF).

Plain Language Summary

In this study, we report a new MICAL‐1 pathogenic variant in a patient without family history for epilepsy, not inherited from his parents. MICAL‐1 is a protein with enzymatic activity that reorganizes the structure of the cell. We proved the pathological effect of this variant by testing its enzymatic activity and found an increase of this activity. This result suggests that non‐familial cases should be tested to find novel pathogenic variants in this gene.

Key points.

• A de novo MICAL‐1 pathogenic variant has been associated with epilepsy with auditory features (EAF).

• MICAL‐1 pathogenic variant can occur in sporadic case with EAF.

• Functional analysis showed that this variant significantly increases MICAL‐1 oxidoreductase activity.

• Screening of sporadic patients is need to determine the precise proportion of case by de novo MICAL‐1 pathogenic variants.

1. INTRODUCTION

Familial epilepsy with auditory features (FEAF), previously known as autosomal‐dominant lateral temporal lobe epilepsy (ADLTE) ¹ is a genetic epilepsy syndrome clinically characterized by focal seizures with auditory or aphasic auras, likely originating from the lateral region of the temporal lobe and onset is in adolescence or early adulthood. ² , ³ Patients show a normal neurological status and negative conventional magnetic resonance imaging (MRI). FEAF is inherited with an autosomal‐dominant pattern with reduced penetrance (70%) and may account for up to 19% of familial focal epilepsies. ³ Earlier, we and others identified variants causing FEAF in the Leucine‐rich Glioma Inactivated 1(LGI1) gene, whose pathogenic variants account for around 30% of FEAF families and 2% of sporadic cases. ³ , ⁴ More recently two further genes, RELN (Reelin) ⁵ and MICAL‐1, ⁶ have been implicated in the etiology of FEAF and altogether may explain an additional 30% of familiar cases. Molecule Interacting with CasL 1 (MICAL‐1) encodes an intracellular protein with oxidoreductase activity implicated in the control of actin cytoskeleton dynamics ⁶ and this enzymatic activity is autoregulated by its C‐terminal domain. Here, we describe a de novo MICAL‐1 pathogenic variant in a sporadic patient with epilepsy with auditory features (EAF), and provide experimental evidence of its effect on MICAL‐1 protein activity, supporting a gain‐of‐function effect underlying MICAL‐1‐mediated pathogenesis of epilepsy.

2. MATERIALS AND METHODS

2.1. Clinical data

Our proband was subjected to an Next Generation Sequencing (NGS) analysis for the presence of epilepsy with auditory features with unknown etiology.

2.2. Genetic data

Peripheral blood samples were collected from the patient and his parents after obtaining written informed consent. DNA was analyzed by NGS with a custom panel of 190 genes associated to epilepsy (Agilent Technologies, Santa Clara, CA) (see gene list in Table S1) using a Sure Select QXT Target enrichment kit (Agilent). Libraries were sequenced on an Illumina Next Seq 500 with 150 bp paired‐ends reads and a high‐output cartridge (Illumina, San Diego, CA). Variants were annotated using ANNOVAR (www.openbioinformatics.org/annovar); all rare (allele frequency < 0.01) exonic variants of unknown significance were verified with Sanger sequencing on the patient and his parents.

2.3. Generation of MICAL‐1 mutant expression construct and enzyme activity test

Mutant expression constructs were generated by site‐directed mutagenesis in plasmids containing human or mouse MICAL‐1 cDNAs using the QuickChange II mutagenesis kit (Agilent Technologies). The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, Carlsbad, CA) was used to measure hydrogen peroxide production from lysates of transfected HEK293T cells, as described previously. ⁷ Briefly, cells transfected with expression plasmids encoding wild‐type or mutant MICAL‐1 were grown for 48 h and lysed by sonication. Reactions were performed in 100 μL consisting of 50 μL of cell lysate, 200 μM of NADPH, 10 μM of Amplex Red Reagent (10‐acetyl‐3,7‐dihydroxyphenoxazine), 100 μU horseradish peroxidase in 20 mM of HEPES (pH 7.4) and 100 mM NaCl. Absorbance at 560 nm was measured using a VectorX3 96‐well plate spectrophotometer (PerkinElmer, Boston, MA). Statistical analyses were performed using Student's t test on six independent experiments. p values significance between groups was established at p ≤ 0.01.

3. RESULTS

3.1. Clinical findings

The proband is a 21‐year‐old right‐handed male with no family history of epilepsy. There were no antecedent illnesses or injuries to the central nervous system and the child had normal psychomotor development. At 14 years and 8 months he presented the first focal seizure with bilateral tonic–clonic evolution, lasting about 2 min. Seizure onset was characterized by an auditory aura defined by feeling of distortion/attenuation of sounds followed by dizziness and aphasia. From 14 years and 5 months he presented on a monthly basis four other seizures with similar semiology and only in one case with bilateral tonic–clonic evolution. Focal seizures with impaired awareness lasted <1 min. The post‐ictal phase, lasting several minutes, was characterized by psychomotor agitation, non‐contextual sentences and difficulty in naming objects.

Numerous video‐EEGs during wakefulness and sleep show a good organization of background activity and the presence of rare sharp waves over the left temporal region. Brain MRI shows absence of structural brain lesions.

After the fifth seizure at 15 years and 8 months, low‐dose carbamazepine (400 mg in the evening) was introduced with seizure remission. At the following yearly follow‐up, the patient reported only the presence of monthly episodes characterized by an unpleasant auditory aura associated with a feeling of fear. These episodes happen more easily in very noisy and crowded places.

The patient underwent two neuropsychological evaluations, the first at the age of 15 years and 8 months, before the introduction of carbamazepine, and the second during the last follow‐up at the age of 21. Table 1 shows the WISC IV sub test scores with evidence of greater logical skills of visual–spatial nature.

TABLE 1.

WISC IV sub test scores.

	15 years	21 years
Verbal comprehension (ICV)	110	112
Visual–spatial reasoning (IRP)	121	122
Working memory (IML)	110	109
Processing speed (IVE)	104	103
Total IQ	115	116

The neuropsychological functions investigated (verbal and visuo‐spatial memory, verbal fluency, attention, executive functions and praxis) were normal. Quality of life measured with the Qolie 31 is normal in all domains. Behavioral aspects (measured with MMPI‐2 and MCMI‐III) are devoid of significant psychopathological connotations.

3.2. Variant features

The MICAL‐1 variant was found by NGS screening with a panel of epilepsy‐related genes, together with additional variations of unknown significance in SCN10A, PTPN23, NPRL3, CACNA1H, KANSL1, all inherited from a parent. Instead the MICAL‐1 variant is present only in the proband (de novo) (Figure 1A).

FIGURE 1.

Pedigree of the family and MICAL‐1 oxidoreductase activity assay. (A) Pedigree of the affected child with EAF pathogenic variant, chromatograms of the proband and his parents are shown. Mutated nucleotide is highlighted and indicated by arrow. (B) MICAL‐1 oxidoreductase activity assay. Measurement of H₂O₂ production from lysates of HEK293T cells transfected with the indicated MICAL‐1 constructs in the presence of 200 μM of NADPH. The mouse (p.Arg902Cys) and human (p.Arg915Cys) pathogenic variants significantly increase the MO domain‐mediated enzymatic activity compared to the corresponding wild‐type proteins, hMICAL‐1 and mMICAL‐1. Representative immunoblots of lysates used for enzymatic reactions are shown below the graphs, **p < 0.01.

This sequence change (NM_022765.4:c.2743C > T) replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 915 of the MICAL‐1 protein (NP_073602.3:p.Arg915Cys). This variant is present in population databases (rs750057550, gnomAD 0.0032%). It has not been reported in the literature in individuals affected with MICAL‐1‐related conditions. Algorithms developed to predict the effect of missense changes on protein structure and function substantially agree on the potential impact of this missense change (Table 2). The Arg915 residue, which lies in the polypeptide linker between the prolin‐rich PPKPP region and C‐terminal domain, is highly conserved across vertebrate species in MICAL‐1 proteins (Figure S1), suggesting that this residue is essential for the structure or function of the link domain in this protein. Finally, the human Arg915 residue corresponds to the mouse Arg902.

TABLE 2.

Pathogenic effect prediction of mutated protein by the main tools.

Engine	Prediction	Score
CADD	Deleterious	31
M‐CAP	Possibly pathogenic	0.063
Mutation tester	Causing disease	0.99
SIFT	Deleterious	0
Poliphen2	Possibly damaging	0.79
Mutation assessor	Uncertain	2.36

3.3. Enzymatic assay

To determine the effects of the p.Arg915Cys variant on MICAL‐1 redox activity, we used an indirect enzyme‐linked assay that measures H₂O₂ production. ⁷ , ⁸ MICAL‐1 mutant or wild‐type proteins were overexpressed in HEK293T cells and their enzymatic activity was measured in cell lysates, as described. ⁷ We found that the oxidoreductase activities of human (hMICAL‐1Arg915Cys) and mouse (mMICAL‐1Arg902Cys) mutants were significantly increased (2,3‐fold and 1,7‐fold p ≤ 0.01, respectively), compared to those of the wild‐type protein (Figure 1B). These results strongly support a pathogenic role for this pathogenic variant.

4. DISCUSSION

In this study, we identified a disease‐linked de novo MICAL‐1 pathogenic variant (p.Arg915Cys) in one sporadic EAF patient and showed that this variant causes a significant increase of MICAL‐1 oxidoreductase activity. The clinical characteristics are similar to those of patients carrying pathogenic variants in LGI1 or RELN. MICAL‐1 regulates the organization of actin cytoskeleton in the nervous system and in a variety of other cells and tissues ⁸ , ⁹ through the redox activity of its MO domain, which induces oxidation of specific amino acid residues of F‐actin, ultimately leading to actin filament disassembly. ¹⁰ , ¹¹ MICAL‐1 oxidoreductase activity is autoinhibited by the C‐terminal CC domain ⁷ so that the wild‐type protein is nearly inactive. This autoinhibition is mediated by binding of the CC domain to the MO domain (see schematic in Figure S1). We studied the functional consequences of the p.Arg915Cys pathogenic variant in HEK293T transfected cells. Our study showed that the p.Arg915Cys pathogenic variant significantly increases the oxidoreductase activity of the protein, suggesting that a gain of function underlies the pathogenesis of MICAL‐1‐mediated epilepsy, as shown previously for other pathogenic variants. ⁶

The p.Arg915Cys pathogenic variant is located in the linker region that connects the prolin‐rich PPKPP region with the C‐terminal domain, which serves an essential function in regulating the enzymatic activity of MICAL‐1. Based on our biochemical results, we suggest that this linker region may also take part in the autoinhibition mechanism of MICAL‐1 oxidoreductase activity.

5. CONCLUSION

The case presented here provides evidence that de novo MICAL‐1 pathogenic variants can occur in sporadic EAF cases. Screening of additional sporadic patients is needed to determine the precise proportion of cases caused by de novo MICAL‐1 pathogenic variants and for genetic counseling purposes.

AUTHOR CONTRIBUTIONS

ED and CN designed the study; PB and RM collected clinical data; RG collected NGS data; ED collected experimental data. PB, CN, and ED wrote the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.

CONFLICT OF INTEREST STATEMENT

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Supporting information

Figure S1

Table S1

Bonanni P, Giorda R, Michelucci R, Nobile C, Dazzo E. A de novo pathogenic variant in MICAL‐1 causes epilepsy with auditory features. Epilepsia Open. 2024;9:1083–1087. 10.1002/epi4.12937